Compositions for modulating SOD-1 expression

ABSTRACT

Disclosed herein are antisense compounds and methods for decreasing SOD-1 mRNA and protein expression. Such methods, compounds, and compositions are useful to treat, prevent, or ameliorate SOD-1 associated diseases, disorders, and conditions. Such SOD-1 associated diseases include amyotrophic sclerosis (ALS).

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledBIOL0240USASEQ_ST25.pdf created Sep. 28, 2016, which is 320 Kb in size.The information in the electronic format of the sequence listing isincorporated herein by reference in its entirety.

FIELD

Provided are compositions and methods for reducing expression ofsuperoxide dismutase 1, soluble (SOD-1) mRNA and protein in an animal.Such methods are useful to treat, prevent, or ameliorateneurodegenerative diseases, including amyotrophic lateral sclerosis(ALS) by inhibiting expression of SOD-1 in an animal.

BACKGROUND

The soluble SOD-1 enzyme (also known as Cu/Zn superoxide dismutase) isone of the superoxide dismutases that provide defense against oxidativedamage of biomolecules by catalyzing the dismutation of superoxide tohydrogen peroxide (H2O2) (Fridovich, Annu. Rev. Biochem., 1995, 64,97-112). The superoxide anion (O2-) is a potentially harmful cellularby-product produced primarily by errors of oxidative phosphorylation inmitochondria (Turrens, J. Physiol. 2003, 552, 335-344)

Mutations in the SOD-1 gene are associated with a dominantly-inheritedform of amyotrophic lateral sclerosis (ALS, also known as Lou Gehrig'sdisease) a disorder characterized by a selective degeneration of upperand lower motor neurons (Rowland, N. Engl. J. Med. 2001, 344,1688-1700). There is a tight genetic linkage between familial ALS andmissense mutations in the SOD1 gene (Rosen, Nature, 1993, 362, 59-62).The toxicity of mutant SOD1 is believed to arise from an initialmisfolding (gain of function) reducing nuclear protection from theactive enzyme (loss of function in the nuclei), a process that may beinvolved in ALS pathogenesis (Sau, Hum. Mol. Genet. 2007, 16,1604-1618).

ALS is a devastating progressive neurodegenerative disease affecting asmany as 30,000 Americans at any given time. The progressive degenerationof the motor neurons in ALS eventually leads to their death. When themotor neurons die, the ability of the brain to initiate and controlmuscle movement is lost. With voluntary muscle action progressivelyaffected, patients in the later stages of the disease may become totallyparalyzed.

Currently lacking are acceptable options for treating suchneurodegenerative diseases. It is therefore an object herein to providemethods for the treatment of such diseases.

SUMMARY

Provided herein are methods, compounds, and compositions for modulatingexpression of superoxide dismutase 1, soluble (SOD-1) mRNA and protein.In certain embodiments, compounds useful for modulating expression ofSOD-1 mRNA and protein are antisense compounds. In certain embodiments,the antisense compounds are modified oligonucleotides.

In certain embodiments, modulation can occur in a cell or tissue. Incertain embodiments, the cell or tissue is in an animal. In certainembodiments, the animal is a human. In certain embodiments, SOD-1 mRNAlevels are reduced. In certain embodiments, SOD-1 protein levels arereduced. Such reduction can occur in a time-dependent manner or in adose-dependent manner.

Also provided are methods, compounds, and compositions useful forpreventing, treating, and ameliorating diseases, disorders, andconditions. In certain embodiments, such SOD-1 related diseases,disorders, and conditions are neurodegenerative diseases. In certainembodiments, such neurodegenerative diseases, disorders, and conditionsinclude amyotrophic lateral sclerosis (ALS).

Such diseases, disorders, and conditions can have one or more riskfactors, causes, or outcomes in common. Certain risk factors and causesfor development of ALS include growing older, having a personal orfamily history, or genetic predisposition. However, the majority of ALScases are sporadic and no known risk factors are known. Certain symptomsand outcomes associated with development of ALS include but are notlimited to: fasciculations, cramps, tight and stiff muscles(spasticity), muscle weakness affecting an arm or a leg, slurred andnasal speech, difficulty walking, difficulty chewing or swallowing(dysphagia), difficulty speaking or forming words (dysarthria), weaknessor atrophy, spasticity, exaggerated reflexes (hyperreflexia), andpresence of Babinski's sign. As ALS progresses, symptoms and outcomes byinclude weakening of other limbs, perhaps accompanied by twitching,muscle cramping, and exaggerated, faster reflexes; problems withchewing, swallowing, and breathing; drooling may occur; eventualparalysis; and death.

In certain embodiments, methods of treatment include administering anSOD-1 antisense compound to an individual in need thereof. In certainembodiments, methods of treatment include administering an SOD-1modified oligonucleotide to an individual in need thereof.

DETAILED DESCRIPTION

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed. Herein, the use ofthe singular includes the plural unless specifically stated otherwise.As used herein, the use of “or” means “and/or” unless stated otherwise.Additionally, as used herein, the use of “and” means “and/or” unlessstated otherwise. Furthermore, the use of the term “including” as wellas other forms, such as “includes” and “included”, is not limiting.Also, terms such as “element” or “component” encompass both elements andcomponents comprising one unit and elements and components that comprisemore than one subunit, unless specifically stated otherwise. Also, allsequences described herein are listed 5′ to 3′, unless otherwise stated.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in this disclosure,including, but not limited to, patents, patent applications, publishedpatent applications, articles, books, treatises, and GENBANK AccessionNumbers and associated sequence information obtainable through databasessuch as National Center for Biotechnology Information (NCBI) and otherdata referred to throughout in the disclosure herein are herebyexpressly incorporated by reference for the portions of the documentdiscussed herein, as well as in their entirety.

Definitions

Unless specific definitions are provided, the nomenclature utilized inconnection with, and the procedures and techniques of, analyticalchemistry, synthetic organic chemistry, and medicinal and pharmaceuticalchemistry described herein are those well known and commonly used in theart. Standard techniques may be used for chemical synthesis, andchemical analysis.

Unless otherwise indicated, the following terms have the followingmeanings:

“2′-deoxynucleoside” (also 2′-deoxyribonucleoside) means a nucleosidecomprising 2′-H furanosyl sugar moiety, as found in naturally occurringdeoxyribonucleosides (DNA). In certain embodiments, a 2′-deoxynucleosidemay comprise a modified nucleobase or may comprise an RNA nucleobase(e.g., uracil).

“2′-deoxyribose sugar” means a 2′-H furanosyl sugar moiety, as found innaturally occurring deoxyribonucleic acids (DNA).

“2′-O-methoxyethyl” (also 2′-MOE and 2′-OCH₂CH₂—OCH₃ and MOE and2′-O-methoxyethylribose) refers to an O-methoxy-ethyl modification ofthe 2′ position of a furanose ring. A 2′-O-methoxyethylribose modifiedsugar is a modified sugar.

“2′-O-methoxyethylribose modified nucleoside” (also 2′-MOE nucleoside)means a nucleoside comprising a 2′-MOE modified sugar moiety.

“2′-substituted nucleoside” means a nucleoside comprising a substituentat the 2′-position of the furanose ring other than H or OH. In certainembodiments, 2′ substituted nucleosides include nucleosides withbicyclic sugar modifications.

“5-methylcytosine” means a cytosine modified with a methyl groupattached to the 5 position. A 5-methylcytosine is a modified nucleobase.

“About” means within ±10% of a value. For example, if it is stated, “thecompounds affected at least about 50% inhibition of SOD-1”, it isimplied that the SOD-1 levels are inhibited within a range of 45% and55%. “Administered concomitantly” refers to the co-administration of twopharmaceutical agents in any manner in which the pharmacological effectsof both are manifest in the patient at the same time. Concomitantadministration does not require that both pharmaceutical agents beadministered in a single pharmaceutical composition, in the same dosageform, or by the same route of administration. The effects of bothpharmaceutical agents need not manifest themselves at the same time. Theeffects need only be overlapping for a period of time and need not becoextensive.

“Administering” means providing a pharmaceutical agent to an animal, andincludes, but is not limited to administering by a medical professionaland self-administering.

“Amelioration” refers to a lessening, slowing, stopping, or reversing ofat least one indicator of the severity of a condition or disease. Theseverity of indicators may be determined by subjective or objectivemeasures, which are known to those skilled in the art.

“Animal” refers to a human or non-human animal, including, but notlimited to, mice, rats, rabbits, dogs, cats, pigs, and non-humanprimates, including, but not limited to, monkeys and chimpanzees.

“Antibody” refers to a molecule characterized by reacting specificallywith an antigen in some way, where the antibody and the antigen are eachdefined in terms of the other. Antibody may refer to a complete antibodymolecule or any fragment or region thereof, such as the heavy chain, thelight chain, Fab region, and Fc region.

“Antisense activity” means any detectable or measurable activityattributable to the hybridization of an antisense compound to its targetnucleic acid. In certain embodiments, antisense activity is a decreasein the amount or expression of a target nucleic acid or protein encodedby such target nucleic acid.

“Antisense compound” means an oligomeric compound that is capable ofundergoing hybridization to a target nucleic acid through hydrogenbonding. Examples of antisense compounds include single-stranded anddouble-stranded compounds, such as, antisense oligonucleotides, siRNAs,shRNAs, ssRNAs, and occupancy-based compounds.

“Antisense inhibition” means reduction of target nucleic acid levels inthe presence of an antisense compound complementary to a target nucleicacid compared to target nucleic acid levels or in the absence of theantisense compound.

“Antisense mechanisms” are all those mechanisms involving hybridizationof a compound with a target nucleic acid, wherein the outcome or effectof the hybridization is either target degradation or target occupancywith concomitant stalling of the cellular machinery involving, forexample, transcription or splicing.

“Antisense oligonucleotide” means a single-stranded oligonucleotidehaving a nucleobase sequence that permits hybridization to acorresponding segment of a target nucleic acid.

“Base complementarity” refers to the capacity for the precise basepairing of nucleobases of an oligonucleotide with correspondingnucleobases in a target nucleic acid (i.e., hybridization), and ismediated by Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogenbinding between corresponding nucleobases.

“Bicyclic sugar” means a furanose ring modified by the bridging of twoatoms. A bicyclic sugar is a modified sugar.

“Bicyclic nucleic acid” or “BNA” refers to a nucleoside or nucleotidewherein the furanose portion of the nucleoside or nucleotide includes abridge connecting two carbon atoms on the furanose ring, thereby forminga bicyclic ring system.

“Cap structure” or “terminal cap moiety” means chemical modifications,which have been incorporated at either terminus of an antisensecompound.

“cEt” or “constrained ethyl” or “cEt modified sugar” means a bicyclicnucleoside having a sugar moiety comprising a bridge connecting the4′-carbon and the 2′-carbon, wherein the bridge has the formula:4′-CH(CH₃)—O-2′. A cEt modified sugar is a modified sugar.

“cEt modified nucleoside” means a bicyclic nucleoside having a sugarmoiety comprising a bridge connecting the 4′-carbon and the 2′-carbon,wherein the bridge has the formula: 4′-CH(CH₃)—O-2′. A cEt modifiedsugar is a modified sugar.

“Chemically distinct region” refers to a region of an antisense compoundthat is in some way chemically different than another region of the sameantisense compound. For example, a region having 2′-O-methoxyethylnucleosides is chemically distinct from a region having nucleosideswithout 2′-O-methoxyethyl modifications.

“Chimeric antisense compound” means an antisense compound that has atleast two chemically distinct regions, each position having a pluralityof subunits.

“Co-administration” means administration of two or more pharmaceuticalagents to an individual. The two or more pharmaceutical agents may be ina single pharmaceutical composition, or may be in separatepharmaceutical compositions. Each of the two or more pharmaceuticalagents may be administered through the same or different routes ofadministration. Co-administration encompasses parallel or sequentialadministration.

“Complementarity” means the capacity for pairing between nucleobases ofa first nucleic acid and a second nucleic acid.

“Comprise,” “comprises,” and “comprising” will be understood to implythe inclusion of a stated step or element or group of steps or elementsbut not the exclusion of any other step or element or group of steps orelements.

“Contiguous nucleobases” means nucleobases immediately adjacent to eachother.

“Designing” or “designed to” refer to the process of designing anoligomeric compound that specifically hybridizes with a selected nucleicacid molecule.

“Diluent” means an ingredient in a composition that lackspharmacological activity, but is pharmaceutically necessary ordesirable. For example, in drugs that are injected, the diluent may be aliquid, e.g. saline solution.

“Dose” means a specified quantity of a pharmaceutical agent provided ina single administration, or in a specified time period. In certainembodiments, a dose may be administered in one, two, or more boluses,tablets, or injections. For example, in certain embodiments wheresubcutaneous administration is desired, the desired dose requires avolume not easily accommodated by a single injection, therefore, two ormore injections may be used to achieve the desired dose. In certainembodiments, the pharmaceutical agent is administered by infusion overan extended period of time or continuously. Doses may be stated as theamount of pharmaceutical agent per hour, day, week, or month.

“Effective amount” in the context of modulating an activity or oftreating or preventing a condition means the administration of thatamount of pharmaceutical agent to a subject in need of such modulation,treatment, or prophylaxis, either in a single dose or as part of aseries, that is effective for modulation of that effect, or fortreatment or prophylaxis or improvement of that condition. The effectiveamount may vary among individuals depending on the health and physicalcondition of the individual to be treated, the taxonomic group of theindividuals to be treated, the formulation of the composition,assessment of the individual's medical condition, and other relevantfactors.

“Efficacy” means the ability to produce a desired effect.

“Expression” includes all the functions by which a gene's codedinformation is converted into structures present and operating in acell. Such structures include, but are not limited to the products oftranscription and translation.

“Fully complementary” or “100% complementary” means each nucleobase of afirst nucleic acid has a complementary nucleobase in a second nucleicacid. In certain embodiments, a first nucleic acid is an antisensecompound and a target nucleic acid is a second nucleic acid.

“Gapmer” means a chimeric antisense compound in which an internal regionhaving a plurality of nucleosides that support RNase H cleavage ispositioned between external regions having one or more nucleosides,wherein the nucleosides comprising the internal region are chemicallydistinct from the nucleoside or nucleosides comprising the externalregions. The internal region may be referred to as a “gap” and theexternal regions may be referred to as the “wings.”

“Gap-narrowed” means a chimeric antisense compound having a gap segmentof 9 or fewer contiguous 2′-deoxyribonucleosides positioned between andimmediately adjacent to 5′ and 3′ wing segments having from 1 to 6nucleosides.

“Gap-widened” means a chimeric antisense compound having a gap segmentof 12 or more contiguous 2′-deoxyribonucleosides positioned between andimmediately adjacent to 5′ and 3′ wing segments having from 1 to 6nucleosides.

“Hybridization” means the annealing of complementary nucleic acidmolecules. In certain embodiments, complementary nucleic acid moleculesinclude, but are not limited to, an antisense compound and a targetnucleic acid. In certain embodiments, complementary nucleic acidmolecules include, but are not limited to, an oligonucleotide and anucleic acid target.

“Identifying an animal having a SOD-1 associated disease” meansidentifying an animal having been diagnosed with a SOD-1 associateddisease or predisposed to develop a SOD-1 associated disease.Individuals predisposed to develop a SOD-1 associated disease includethose having one or more risk factors for developing a SOD-1 associateddisease, including, growing older, having a personal or family history,or genetic predisposition of one or more SOD-1 associated diseases. Suchidentification may be accomplished by any method including evaluating anindividual's medical history and standard clinical tests or assessments,such as genetic testing.

“Immediately adjacent” means there are no intervening elements betweenthe immediately adjacent elements.

“Individual” means a human or non-human animal selected for treatment ortherapy.

“Inhibiting SOD-1” means reducing the level or expression of a SOD-1mRNA and/or protein. In certain embodiments, SOD-1 mRNA and/or proteinlevels are inhibited in the presence of an antisense compound targetingSOD-1, including a modified oligonucleotide targeting SOD-1, as comparedto expression of SOD-1 mRNA and/or protein levels in the absence of aSOD-1 antisense compound, such as a modified oligonucleotide.

“Inhibiting the expression or activity” refers to a reduction orblockade of the expression or activity and does not necessarily indicatea total elimination of expression or activity.

“Internucleoside linkage” refers to the chemical bond betweennucleosides.

“Linked nucleosides” means adjacent nucleosides linked together by aninternucleoside linkage.

“Mismatch” or “non-complementary nucleobase” refers to the case when anucleobase of a first nucleic acid is not capable of pairing with thecorresponding nucleobase of a second or target nucleic acid.

“Mixed backbone” means a pattern of internucleoside linkages includingat least two different internucleoside linkages. For example, anoligonucleotide with a mixed backbone may include at least onephosphodiester linkage and at least one phosphorothioate linkage.

“Modified internucleoside linkage” refers to a substitution or anychange from a naturally occurring internucleoside bond (i.e., aphosphodiester internucleoside bond).

“Modified nucleobase” means any nucleobase other than adenine, cytosine,guanine, thymidine, or uracil. An “unmodified nucleobase” means thepurine bases adenine (A) and guanine (G), and the pyrimidine basesthymine (T), cytosine (C), and uracil (U).

A “modified nucleoside” means a nucleoside having, independently, amodified sugar moiety and/or modified nucleobase.

“Modified nucleotide” means a nucleotide having, independently, amodified sugar moiety, modified internucleoside linkage, and/or modifiednucleobase.

“Modified oligonucleotide” means an oligonucleotide comprising at leastone modified internucleoside linkage, modified sugar, and/or modifiednucleobase.

“Modified sugar” means substitution and/or any change from a naturalsugar moiety.

“Monomer” means a single unit of an oligomer. Monomers include, but arenot limited to, nucleosides and nucleotides, whether naturally occurringor modified.

“Motif” means the pattern of unmodified and modified nucleosides in anantisense compound.

“Natural sugar moiety” means a sugar moiety found in DNA (2′-H) or RNA(2′-OH).

“Naturally occurring internucleoside linkage” means a 3′ to 5′phosphodiester linkage.

“Non-complementary nucleobase” refers to a pair of nucleobases that donot form hydrogen bonds with one another or otherwise supporthybridization.

“Nucleic acid” refers to molecules composed of monomeric nucleotides. Anucleic acid includes, but is not limited to, ribonucleic acids (RNA),deoxyribonucleic acids (DNA), single-stranded nucleic acids,double-stranded nucleic acids, small interfering ribonucleic acids(siRNA), and microRNAs (miRNA).

“Nucleobase” means a heterocyclic moiety capable of pairing with a baseof another nucleic acid.

“Nucleobase complementarity” refers to a nucleobase that is capable ofbase pairing with another nucleobase. For example, in DNA, adenine (A)is complementary to thymine (T). For example, in RNA, adenine (A) iscomplementary to uracil (U). In certain embodiments, complementarynucleobase refers to a nucleobase of an antisense compound that iscapable of base pairing with a nucleobase of its target nucleic acid.For example, if a nucleobase at a certain position of an antisensecompound is capable of hydrogen bonding with a nucleobase at a certainposition of a target nucleic acid, then the position of hydrogen bondingbetween the oligonucleotide and the target nucleic acid is considered tobe complementary at that nucleobase pair.

“Nucleobase sequence” means the order of contiguous nucleobasesindependent of any sugar, linkage, and/or nucleobase modification.

“Nucleoside” means a nucleobase linked to a sugar.

“Nucleoside mimetic” includes those structures used to replace the sugaror the sugar and the base and not necessarily the linkage at one or morepositions of an oligomeric compound such as for example nucleosidemimetics having morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranyl,bicyclo, or tricyclo sugar mimetics, e.g., non furanose sugar units.Nucleotide mimetic includes those structures used to replace thenucleoside and the linkage at one or more positions of an oligomericcompound such as for example peptide nucleic acids or morpholinos(morpholinos linked by —N(H)—C(═O)—O— or other non-phosphodiesterlinkage). Sugar surrogate overlaps with the slightly broader termnucleoside mimetic but is intended to indicate replacement of the sugarunit (furanose ring) only. The tetrahydropyranyl rings provided hereinare illustrative of an example of a sugar surrogate wherein the furanosesugar group has been replaced with a tetrahydropyranyl ring system.“Mimetic” refers to groups that are substituted for a sugar, anucleobase, and/or internucleoside linkage. Generally, a mimetic is usedin place of the sugar or sugar-internucleoside linkage combination, andthe nucleobase is maintained for hybridization to a selected target.

“Nucleotide” means a nucleoside having a phosphate group covalentlylinked to the sugar portion of the nucleoside.

“Off-target effect” refers to an unwanted or deleterious biologicaleffect associated with modulation of RNA or protein expression of a geneother than the intended target nucleic acid.

“Oligomeric compound” or “oligomer” means a polymer of linked monomericsubunits which is capable of hybridizing to at least a region of anucleic acid molecule.

“Oligonucleotide” means a polymer of linked nucleosides each of whichcan be modified or unmodified, independent one from another.

“Parenteral administration” means administration through injection(e.g., bolus injection) or infusion. Parenteral administration includessubcutaneous administration, intravenous administration, intramuscularadministration, intraarterial administration, intraperitonealadministration, or intracranial administration, e.g., intrathecal orintracerebroventricular administration.

“Peptide” means a molecule formed by linking at least two amino acids byamide bonds. Without limitation, as used herein, peptide refers topolypeptides and proteins.

“Pharmaceutical agent” means a substance that provides a therapeuticbenefit when administered to an individual. For example, in certainembodiments, a modified oligonucleotide targeted to SOD-1 is apharmaceutical agent.

“Pharmaceutical composition” means a mixture of substances suitable foradministering to a subject. For example, a pharmaceutical compositionmay comprise a modified oligonucleotide and a sterile aqueous solution.

“Pharmaceutically acceptable derivative” encompasses pharmaceuticallyacceptable salts, conjugates, prodrugs or isomers of the compoundsdescribed herein.

“Pharmaceutically acceptable salts” means physiologically andpharmaceutically acceptable salts of antisense compounds, i.e., saltsthat retain the desired biological activity of the parentoligonucleotide and do not impart undesired toxicological effectsthereto.

“Phosphorothioate linkage” means a linkage between nucleosides where thephosphodiester bond is modified by replacing one of the non-bridgingoxygen atoms with a sulfur atom. A phosphorothioate linkage is amodified internucleoside linkage.

“Portion” means a defined number of contiguous (i.e., linked)nucleobases of a nucleic acid. In certain embodiments, a portion is adefined number of contiguous nucleobases of a target nucleic acid. Incertain embodiments, a portion is a defined number of contiguousnucleobases of an antisense compound.

“Prevent” or “preventing” refers to delaying or forestalling the onsetor development of a disease, disorder, or condition for a period of timefrom minutes to days, weeks to months, or indefinitely.

“Prodrug” means a therapeutic agent that is prepared in an inactive formthat is converted to an active form (i.e., drug) within the body orcells thereof by the action of endogenous enzymes or other chemicalsand/or conditions.

“Prophylactically effective amount” refers to an amount of apharmaceutical agent that provides a prophylactic or preventativebenefit to an animal.

“Region” is defined as a portion of the target nucleic acid having atleast one identifiable structure, function, or characteristic.

“Ribonucleotide” means a nucleotide having a hydroxy at the 2′ positionof the sugar portion of the nucleotide. Ribonucleotides may be modifiedwith any of a variety of substituents.

“Salts” mean a physiologically and pharmaceutically acceptable salts ofantisense compounds, i.e., salts that retain the desired biologicalactivity of the parent oligonucleotide and do not impart undesiredtoxicological effects thereto.

“Segments” are defined as smaller or sub-portions of regions within atarget nucleic acid.

“Shortened” or “truncated” versions of oligonucleotides SOD-1ght hereinhave one, two or more nucleosides deleted.

“Side effects” means physiological responses attributable to a treatmentother than desired effects. In certain embodiments, side effectsinclude, without limitation, injection site reactions, liver functiontest abnormalities, renal function abnormalities, liver toxicity, renaltoxicity, central nervous system abnormalities, and myopathies.

“Single-stranded oligonucleotide” means an oligonucleotide which is nothybridized to a complementary strand.

“Sites,” as used herein, are defined as unique nucleobase positionswithin a target nucleic acid.

“Slows progression” means decrease in the development of the disease.

“SOD-1” means the mammalian gene superoxide dismutase 1, soluble(SOD-1), including the human gene superoxide dismutase 1, soluble(SOD-1).

“SOD-1 associated disease” means any disease associated with any SOD-1nucleic acid or expression product thereof. Such diseases may include aneurodegenerative disease. Such neurodegenerative diseases may includeamyotrophic lateral sclerosis (ALS).

“SOD-1 mRNA” means any messenger RNA expression product of a DNAsequence encoding SOD-1.

“SOD-1 nucleic acid” means any nucleic acid encoding SOD-1. For example,in certain embodiments, a SOD-1 nucleic acid includes a DNA sequenceencoding SOD-1, an RNA sequence transcribed from DNA encoding SOD-1(including genomic DNA comprising introns and exons), and a mRNAsequence encoding SOD-1. “SOD-1 mRNA” means a mRNA encoding a SOD-1protein.

“SOD-1 protein” means the polypeptide expression product of a SOD-1nucleic acid.

“Specifically hybridizable” refers to an antisense compound having asufficient degree of complementarity between an oligonucleotide and atarget nucleic acid to induce a desired effect, while exhibiting minimalor no effects on non-target nucleic acids under conditions in whichspecific binding is desired, i.e., under physiological conditions in thecase of in vivo assays and therapeutic treatments.

“Stringent hybridization conditions” or “stringent conditions” refer toconditions under which an oligomeric compound will hybridize to itstarget sequence, but to a minimal number of other sequences.

“Subject” means a human or non-human animal selected for treatment ortherapy.

“Sugar chemistry motif” means a pattern of sugar modifications includingat least two different sugar modifications. For example, anoligonucleotide with a mixed backbone may include at least one2′-O-methoxyethyl modified nucleoside, and/or one cEt modifiednucleoside, and/or one 2′-deoxynucleoside.

“Target” refers to a protein, the modulation of which is desired.

“Target gene” refers to a gene encoding a target.

“Targeting” or “targeted” means the process of design and selection ofan antisense compound that will specifically hybridize to a targetnucleic acid and induce a desired effect.

“Target nucleic acid,” “target RNA,” and “target RNA transcript” and“nucleic acid target” all mean a nucleic acid capable of being targetedby antisense compounds.

“Target region” means a portion of a target nucleic acid to which one ormore antisense compounds is targeted.

“Target segment” means the sequence of nucleotides of a target nucleicacid to which an antisense compound is targeted. “5′ target site” refersto the 5′-most nucleotide of a target segment. “3′ target site” refersto the 3′-most nucleotide of a target segment.

“Therapeutically effective amount” means an amount of a pharmaceuticalagent that provides a therapeutic benefit to an individual.

“Treat” or “treating” or “treatment” refers administering a compositionto effect an alteration or improvement of the disease or condition.

“Unmodified nucleobases” mean the purine bases adenine (A) and guanine(G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).

“Unmodified nucleotide” means a nucleotide composed of naturallyoccurring nucleobases, sugar moieties, and internucleoside linkages. Incertain embodiments, an unmodified nucleotide is an RNA nucleotide (i.e.β-D-ribonucleosides) or a DNA nucleotide (i.e. β-D-deoxyribonucleoside).

“Wing segment” means a plurality of nucleosides modified to impart to anoligonucleotide properties such as enhanced inhibitory activity,increased binding affinity for a target nucleic acid, or resistance todegradation by in vivo nucleases.

CERTAIN EMBODIMENTS

Certain embodiments provide methods, compounds, and compositions forinhibiting SOD-1 mRNA and protein expression. Certain embodimentsprovide methods, compounds, and composition for decreasing SOD-1 mRNAand protein levels.

Certain embodiments provide antisense compounds targeted to a SOD-1nucleic acid. In certain embodiments, the SOD-1 nucleic acid is thesequence set forth in GENBANK Accession No. NM_000454.4 (incorporatedherein as SEQ ID NO: 1), GENBANK Accession No. NT_011512.10 truncatedfrom nucleotides 18693000 to Ser. No. 18/704,000 (incorporated herein asSEQ ID NO: 2), and the complement of GENBANK Accession No.NW_001114168.1 truncated from nucleotides 2258000 to U.S. Pat. No.2,271,000 (incorporated herein as SEQ ID NO: 3).

Certain embodiments provide methods for the treatment, prevention, oramelioration of diseases, disorders, and conditions associated withSOD-1 in an individual in need thereof. Also contemplated are methodsfor the preparation of a medicament for the treatment, prevention, oramelioration of a disease, disorder, or condition associated with SOD-1.SOD-1 associated diseases, disorders, and conditions includeneurodegenerative diseases. In certain embodiments, SOD-1 associateddiseases include amyotrophic lateral sclerosis (ALS).

Embodiment 1

A compound, comprising a modified oligonucleotide consisting of 12 to 30linked nucleosides and having a nucleobase sequence comprising at least8, at least 9, at least 10, at least 11, at least 12, at least 13, atleast 14, at least 15, at least 16, at least 17, at least 18, at least19, or at least 20 consecutive nucleobases of any of the nucleobasesequences of SEQ ID NOs: 118-1461.

Embodiment 2

A compound, comprising a modified oligonucleotide consisting of 12 to 30linked nucleosides and having a nucleobase sequence comprising at least8, at least 9, at least 10, at least 11, at least 12, at least 13, atleast 14, at least 15, at least 16, at least 17, at least 18, at least19, or at least 20 consecutive nucleobases of any of the nucleobasesequences of SEQ ID NOs:15, 21, 23, 47, 54, and 67, wherein at least oneinternucleoside linkage is a phosphodiester linkage.

Embodiment 3

The compound of any preceding embodiment, wherein the modifiedoligonucleotide has a mixed backbone.

Embodiment 4

The compound of embodiment 3, wherein the mixed backbone motif is any ofthe following:

sossssssssoooss, sooossssssssoss, sooosssssssssoss, soosssssssssooss,sooossssssssooss, sooosssssssssooss, sooossssssssssooss,sooosssssssssssooos, soooossssssssssooss, sooosssssssssssooss,sososssssssssssosos, and sooossssssssssoooss, wherein s = aphosphorothioate internucleoside linkage, and o = a phosphodiesterinternucleoside linkage.

Embodiment 5

The compound of any preceding embodiment, wherein the modifiedoligonucleotide has a sugar chemistry motif of any of the following:

ekddddddddekekee, kekeddddddddekek, eeeedddddddddkkee,eeeeddddddddekeke, eeeeddddddddkekee, eeeeddddddddkkeee,eeeeeddddddddkkee, eeeekddddddddkeee, eeeekdddddddkeeee,eeekddddddddkeeee, eeekkdddddddkkeee, eekkdddddddddkkee,eekkddddddddeeeee, eekkddddddddkkeee, ekekddddddddeeeee,ekekddddddddkekee, and kekeddddddddeeeee, wherein e = a2′-O-methoxyethylribose modified sugar, k = a cEt modified sugar, d = a2′-deoxyribose sugar,

Embodiment 6

The compound of any preceding embodiment, wherein the nucleobasesequence of the modified oligonucleotide is at least 80%, at least 81%,at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% complementary to SEQ IDNO: 1 or SEQ ID NO: 2.

Embodiment 7

The compound of any preceding embodiment, consisting of asingle-stranded modified oligonucleotide.

Embodiment 8

The compound of any preceding embodiment, wherein at least oneinternucleoside linkage is a modified internucleoside linkage.

Embodiment 9

The compound of embodiment 8, wherein at least one modifiedinternucleoside linkage is a phosphorothioate internucleoside linkage.

Embodiment 10

The compound of embodiment 9, wherein each modified internucleosidelinkage is a phosphorothioate internucleoside linkage.

Embodiment 11

The compound of any preceding embodiment, wherein at least oneinternucleoside linkage is a phosphodiester internucleoside linkage.

Embodiment 12

The compound of any preceding embodiment, wherein at least oneinternucleoside linkage is a phosphorothioate linkage and at least oneinternucleoside linkage is a phosphodiester linkage.

Embodiment 13

The compound of any preceding embodiment, wherein at least onenucleoside comprises a modified nucleobase.

Embodiment 14

The compound of embodiment 13, wherein the modified nucleobase is a5-methylcytosine.

Embodiment 15

The compound of any preceding embodiment, wherein at least onenucleoside of the modified oligonucleotide comprises a modified sugar.

Embodiment 16

The compound of embodiment 15, wherein the at least one modified sugaris a bicyclic sugar.

Embodiment 17

The compound of embodiment 16, wherein the bicyclic sugar comprises achemical link between the 2′ and 4′ position of the sugar4′-CH₂—N(R)—O-2′ bridge wherein R is, independently, H, C₁-C₁₂ alkyl, ora protecting group.

Embodiment 18

The compound of embodiment 17, wherein the bicyclic sugar comprises a4′-CH₂—N(R)—O-2′ bridge wherein R is, independently, H, C₁-C₁₂ alkyl, ora protecting group.

Embodiment 19

The compound of embodiment 15, wherein at least one modified sugarcomprises a 2′-O-methoxyethyl group.

Embodiment 20

The compound of embodiment 15, wherein the modified sugar comprises a2′-O(CH₂)₂—OCH₃ group.

Embodiment 21

The compound of any preceding embodiment, wherein the modifiedoligonucleotide comprises:

-   -   a gap segment consisting of 10 linked deoxynucleosides;    -   a 5′ wing segment consisting of 5 linked nucleosides; and    -   a 3′ wing segment consisting of 5 linked nucleosides;    -   wherein the gap segment is positioned between the 5′ wing        segment and the 3′ wing segment and wherein each nucleoside of        each wing segment comprises a modified sugar.

Embodiment 22

The compound of any preceding embodiment, wherein the modifiedoligonucleotide comprises:

-   -   a gap segment consisting of 9 linked deoxynucleosides;    -   a 5′ wing segment consisting of 5 linked nucleosides; and    -   a 3′ wing segment consisting of 5 linked nucleosides;    -   wherein the gap segment is positioned between the 5′ wing        segment and the 3′ wing segment and wherein each nucleoside of        each wing segment comprises a modified sugar.

Embodiment 23

The compound of any preceding embodiment, wherein the modifiedoligonucleotide comprises:

-   -   a gap segment consisting of 8 linked deoxynucleosides;    -   a 5′ wing segment consisting of 5 linked nucleosides; and    -   a 3′ wing segment consisting of 5 linked nucleosides;    -   wherein the gap segment is positioned between the 5′ wing        segment and the 3′ wing segment and wherein each nucleoside of        each wing segment comprises a modified sugar.

Embodiment 24

The compound of any preceding embodiment, wherein the modifiedoligonucleotide comprises:

-   -   a gap segment consisting of 8 linked deoxynucleosides;    -   a 5′ wing segment consisting of 4 linked nucleosides; and    -   a 3′ wing segment consisting of 5 linked nucleosides;    -   wherein the gap segment is positioned between the 5′ wing        segment and the 3′ wing segment and wherein each nucleoside of        each wing segment comprises a modified sugar.

Embodiment 25

The compound of any preceding embodiment, wherein the modifiedoligonucleotide comprises:

-   -   a gap segment consisting of 8 linked deoxynucleosides;    -   a 5′ wing segment consisting of 5 linked nucleosides; and    -   a 3′ wing segment consisting of 7 linked nucleosides;    -   wherein the gap segment is positioned between the 5′ wing        segment and the 3′ wing segment and wherein each nucleoside of        each wing segment comprises a modified sugar.

Embodiment 26

The compound of any preceding embodiment, wherein the modifiedoligonucleotide comprises:

-   -   a gap segment consisting of 8 linked deoxynucleosides;    -   a 5′ wing segment consisting of 6 linked nucleosides; and    -   a 3′ wing segment consisting of 6 linked nucleosides;    -   wherein the gap segment is positioned between the 5′ wing        segment and the 3′ wing segment and wherein each nucleoside of        each wing segment comprises a modified sugar.

Embodiment 27

The compound of any preceding embodiment, wherein the modifiedoligonucleotide comprises:

-   -   a gap segment consisting of 9 linked deoxynucleosides;    -   a 5′ wing segment consisting of 6 linked nucleosides; and    -   a 3′ wing segment consisting of 5 linked nucleosides;    -   wherein the gap segment is positioned between the 5′ wing        segment and the 3′ wing segment and wherein each nucleoside of        each wing segment comprises a modified sugar.

Embodiment 28

The compound of any preceding embodiment, wherein the modifiedoligonucleotide consists of 12, 13, 14, 15, 16, 17, 18, 19, or 20 linkednucleosides.

Embodiment 29

A compound consisting of a modified oligonucleotide according to thefollowing formula:

Embodiment 30

A compound consisting of a modified oligonucleotide according to thefollowing formula:

Embodiment 31

A compound consisting of a modified oligonucleotide according to thefollowing formula:

Embodiment 32

A compound consisting of a modified oligonucleotide according to thefollowing formula:

Embodiment 33

A compound consisting of a modified oligonucleotide according to thefollowing formula:

Embodiment 34

A compound consisting of a modified oligonucleotide according to thefollowing formula:

Embodiment 35

A compound consisting of a modified oligonucleotide according to thefollowing formula:

Embodiment 36

A compound consisting of a modified oligonucleotide according to thefollowing formula:

Embodiment 37

A compound consisting of a modified oligonucleotide according to thefollowing formula: mCes Aeo Ges Geo Aes Tds Ads mCds Ads Tds Tds TdsmCds Tds Ads mCeo Aes Geo mCes Te; wherein,

-   -   A=an adenine,    -   mC=a 5′-methylcytosine    -   G=a guanine,    -   T=a thymine,    -   e=a 2′-O-methoxyethylribose modified sugar,    -   d=a 2′-deoxyribose sugar,    -   s=a phosphorothioate internucleoside linkage, and    -   o=a phosphodiester internucleoside linkage.

Embodiment 38

A compound consisting of a modified oligonucleotide according to thefollowing formula: Tes Teo Aeo Aes Tds Gds Tds Tds Tds Ads Tds mCds AkoGko Ges Aes Te; wherein,

-   -   A=an adenine,    -   mC=a 5′-methylcytosine    -   G=a guanine,    -   T=a thymine,    -   e=a 2′-O-methoxyethylribose modified sugar,    -   k=a cEt modified sugar,    -   d=a 2′-deoxyribose sugar,    -   s=a phosphorothioate internucleoside linkage, and    -   o=a phosphodiester internucleoside linkage.

Embodiment 39

A compound consisting of a modified oligonucleotide according to thefollowing formula: Ges Geo Aeo Teo Ads mCds Ads Tds Tds Tds mCds Tds AdsmCko Aks Ges mCe;

-   -   wherein,    -   A=an adenine,    -   mC=a 5′-methylcytosine    -   G=a guanine,    -   T=a thymine,    -   e=a 2′-O-methoxyethylribose modified sugar,    -   k=a cEt modified sugar,    -   d=a 2′-deoxyribose sugar,    -   s=a phosphorothioate internucleoside linkage, and    -   o=a phosphodiester internucleoside linkage.

Embodiment 40

A compound consisting of a modified oligonucleotide according to thefollowing formula: Ges Geo Aeo Teo Aes mCds Ads Tds Tds Tds mCds Tds AdsmCko Aks Ges mCe; wherein,

-   -   A=an adenine,    -   mC=a 5′-methylcytosine    -   G=a guanine,    -   T=a thymine,    -   e=a 2′-O-methoxyethylribose modified sugar,    -   k=a cEt modified sugar,    -   d=a 2′-deoxyribose sugar,    -   s=a phosphorothioate internucleoside linkage, and    -   o=a phosphodiester internucleoside linkage.

Embodiment 41

A compound consisting of a modified oligonucleotide according to thefollowing formula: Ges Geo Aeo Teo Aks mCds Ads Tds Tds Tds mCds Tds AdsmCko Aes Ges mCe; wherein,

-   -   A=an adenine,    -   mC=a 5′-methylcytosine    -   G=a guanine,    -   T=a thymine,    -   e=a 2′-O-methoxyethylribose modified sugar,    -   k=a cEt modified sugar,    -   d=a 2′-deoxyribose sugar,    -   s=a phosphorothioate internucleoside linkage, and    -   o=a phosphodiester internucleoside linkage.

Embodiment 42

A compound consisting of a modified oligonucleotide according to thefollowing formula: Aes Gko Teo Gks Tds Tds Tds Ads Ads Tds Gds Tds TkoTeo Aks Tes mCe; wherein,

-   -   A=an adenine,    -   mC=a 5′-methylcytosine    -   G=a guanine,    -   T=a thymine,    -   e=a 2′-O-methoxyethylribose modified sugar,    -   k=a cEt modified sugar,    -   d=a 2′-deoxyribose sugar,    -   s=a phosphorothioate internucleoside linkage, and    -   o=a phosphodiester internucleoside linkage.

Embodiment 43

A compound consisting of a modified oligonucleotide according to thefollowing formula: Aes Gko Teo Gks Tds Tds Tds Ads Ads Tds Gds Tds TeoTeo Aes Tes mCe; wherein,

-   -   A=an adenine,    -   mC=a 5′-methylcytosine    -   G=a guanine,    -   T=a thymine,    -   e=a 2′-O-methoxyethylribose modified sugar,    -   k=a cEt modified sugar,    -   d=a 2′-deoxyribose sugar,    -   s=a phosphorothioate internucleoside linkage, and    -   o=a phosphodiester internucleoside linkage.

Embodiment 44

A compound consisting of a modified oligonucleotide according to thefollowing formula: Aes Geo Tko Gks Tds Tds Tds Ads Ads Tds Gds Tds TeoTeo Aes Tes mCe; wherein,

-   -   A=an adenine,    -   mC=a 5′-methylcytosine    -   G=a guanine,    -   T=a thymine,    -   e=a 2′-O-methoxyethylribose modified sugar,    -   k=a cEt modified sugar,    -   d=a 2′-deoxyribose sugar,    -   s=a phosphorothioate internucleoside linkage, and    -   o=a phosphodiester internucleoside linkage.

Embodiment 45

A compound consisting of a modified oligonucleotide according to thefollowing formula: mCes mCeo Geo Teo mCeo Gds mCds mCds mCds Tds TdsmCds Ads Gds mCds Aeo mCeo Ges mCes Ae, wherein,

-   -   A=an adenine,    -   mC=a 5′-methylcytosine    -   G=a guanine,    -   T=a thymine,    -   e=a 2′-O-methoxyethylribose modified sugar,    -   d=a 2′-deoxyribose sugar,    -   s=a phosphorothioate internucleoside linkage, and    -   o=a phosphodiester internucleoside linkage.

Embodiment 46

A compound consisting of a modified oligonucleotide according to thefollowing formula: mCes mCeo Geo Teo mCes Gds mCds mCds mCds Tds TdsmCds Ads Ges mCeo Aeo mCeo Ges mCes Ae, wherein,

-   -   A=an adenine,    -   mC=a 5′-methylcytosine    -   G=a guanine,    -   T=a thymine,    -   e=a 2′-O-methoxyethylribose modified sugar,    -   d=a 2′-deoxyribose sugar,    -   s=a phosphorothioate internucleoside linkage, and    -   o=a phosphodiester internucleoside linkage.

Embodiment 47

A compound consisting of a modified oligonucleotide according to thefollowing formula: mCes mCeo Geo Teo mCes Gds mCds mCds mCds Tds TdsmCds Ads Gds mCds Aeo mCeo Geo mCes Ae, wherein,

-   -   A=an adenine,    -   mC=a 5′-methylcytosine    -   G=a guanine,    -   T=a thymine,    -   e=a 2′-O-methoxyethylribose modified sugar,    -   d=a 2′-deoxyribose sugar,    -   s=a phosphorothioate internucleoside linkage, and    -   o=a phosphodiester internucleoside linkage.

Embodiment 48

A compound consisting of a modified oligonucleotide according to thefollowing formula: Aes mCeo Aeo mCeo mCes Tds Tds mCds Ads mCds Tds GdsGds Tds mCds mCeo Aeo Teo Tes Ae, wherein,

-   -   A=an adenine,    -   mC=a 5′-methylcytosine    -   G=a guanine,    -   T=a thymine,    -   e=a 2′-O-methoxyethylribose modified sugar,    -   d=a 2′-deoxyribose sugar,    -   s=a phosphorothioate internucleoside linkage, and    -   o=a phosphodiester internucleoside linkage.

Embodiment 49

A compound consisting of a modified oligonucleotide according to thefollowing formula: Ges Geo mCeo Geo Aes Tds mCds mCds mCds Ads Ads TdsTds Ads mCds Aeo mCeo mCeo Aes mCe, wherein,

-   -   A=an adenine,    -   mC=a 5′-methylcytosine    -   G=a guanine,    -   T=a thymine,    -   e=a 2′-O-methoxyethylribose modified sugar,    -   d=a 2′-deoxyribose sugar,    -   s=a phosphorothioate internucleoside linkage, and    -   o=a phosphodiester internucleoside linkage.

Embodiment 50

A compound consisting of a modified oligonucleotide according to thefollowing formula: Ges Geo mCeo Geo Aes Tes mCds mCds mCds Ads Ads TdsTds Ads mCeo Aeo mCeo mCes Aes mCe, wherein,

-   -   A=an adenine,    -   mC=a 5′-methylcytosine    -   G=a guanine,    -   T=a thymine,    -   e=a 2′-O-methoxyethylribose modified sugar,    -   d=a 2′-deoxyribose sugar,    -   s=a phosphorothioate internucleoside linkage, and    -   o=a phosphodiester internucleoside linkage.

Embodiment 51

A compound consisting of a modified oligonucleotide according to thefollowing formula: Ges Geo mCeo Geo Aes Tds mCds mCds mCds Ads Ads TdsTds Aes mCeo Aeo mCeo mCes Aes mCe, wherein,

-   -   A=an adenine,    -   mC=a 5′-methylcytosine    -   G=a guanine,    -   T=a thymine,    -   e=a 2′-O-methoxyethylribose modified sugar,    -   d=a 2′-deoxyribose sugar,    -   s=a phosphorothioate internucleoside linkage, and    -   o=a phosphodiester internucleoside linkage.

Embodiment 52

A compound consisting of a modified oligonucleotide according to thefollowing formula: Ges Geo mCeo Geo Aeo Tes mCds mCds mCds Ads Ads TdsTds Ads mCds Aeo mCeo mCes Aes mCe, wherein,

-   -   A=an adenine,    -   mC=a 5′-methylcytosine    -   G=a guanine,    -   T=a thymine,    -   e=a 2′-O-methoxyethylribose modified sugar,    -   d=a 2′-deoxyribose sugar,    -   s=a phosphorothioate internucleoside linkage, and    -   o=a phosphodiester internucleoside linkage.

Embodiment 53

A compound consisting of a modified oligonucleotide according to thefollowing formula: Ges Teo mCeo Geo mCes mCds mCds Tds Tds mCds Ads GdsmCds Ads mCds Geo mCeo Aeo mCes Ae, wherein,

-   -   A=an adenine,    -   mC=a 5′-methylcytosine    -   G=a guanine,    -   T=a thymine,    -   e=a 2′-O-methoxyethylribose modified sugar,    -   d=a 2′-deoxyribose sugar,    -   s=a phosphorothioate internucleoside linkage, and    -   o=a phosphodiester internucleoside linkage.

Embodiment 54

A compound consisting of a modified oligonucleotide according to thefollowing formula: Tes mCeo Geo mCeo mCes mCds Tds Tds mCds Ads Gds mCdsAds mCds Gds mCeo Aeo mCeo Aes mCe, wherein,

-   -   A=an adenine,    -   mC=a 5′-methylcytosine    -   G=a guanine,    -   T=a thymine,    -   e=a 2′-O-methoxyethylribose modified sugar,    -   d=a 2′-deoxyribose sugar,    -   s=a phosphorothioate internucleoside linkage, and    -   o=a phosphodiester internucleoside linkage.

Embodiment 55

A compound consisting of a modified oligonucleotide according to thefollowing formula: Ges Aes Aes Aes Tes Tds Gds Ads Tds Gds Ads Tds GdsmCds mCds mCes Tes Ges mCes Ae, wherein,

-   -   A=an adenine,    -   mC=a 5′-methylcytosine    -   G=a guanine,    -   T=a thymine,    -   e=a 2′-O-methoxyethylribose modified sugar,    -   d=a 2′-deoxyribose sugar, and    -   s=a phosphorothioate internucleoside linkage.

Embodiment 56

A composition comprising the compound of any preceding embodiment orsalt thereof and at least one of a pharmaceutically acceptable carrieror diluent.

Embodiment 57

A method comprising administering to an animal the compound orcomposition of any preceding embodiment.

Embodiment 58

The method of embodiment 57, wherein the animal is a human.

Embodiment 59

The method of embodiment 57, wherein administering the compoundprevents, treats, ameliorates, or slows progression of a SOD-1associated disease.

Embodiment 60

The method of embodiment 59, wherein the SOD-1 associated disease is aneurodegenerative disease.

Embodiment 61

The method of embodiment 60, wherein the SOD-1 associated disease isALS.

Embodiment 62

Use of the compound or composition of any preceding embodiment for themanufacture of a medicament for treating a neurodegenerative disorder.

Embodiment 63

Use of the compound or composition of any preceding embodiment for themanufacture of a medicament for treating ALS.

Embodiment 64

The compound or composition of any preceding embodiment wherein themodified oligonucleotide does not have the nucleobase sequence of SEQ IDNO: 21.

Embodiment 65

The compound or composition of any preceding embodiment wherein themodified oligonucleotide does not have the nucleobase sequence of any ofSEQ ID NOs: 21-118.

Embodiment 66

A compound comprising a modified oligonucleotide consisting of 12 to 30linked nucleosides and having a nucleobase sequence, wherein thenucleobase sequence comprises an at least 12 consecutive nucleobaseportion complementary to an equal number of nucleobases of nucleotides665 to 684 of SEQ ID NO: 1, wherein the modified oligonucleotide is atleast 80% complementary to SEQ ID NO: 1.

Embodiment 67

The compound of embodiment 66, wherein the modified oligonucleotide is100% complementary to SEQ ID NO: 1.

Embodiment 68

The compound of embodiment 66, wherein the modified oligonucleotide is asingle-stranded modified oligonucleotide.

Embodiment 69

The compound of embodiments 66-68 wherein at least one internucleosidelinkage is a modified internucleoside linkage.

Embodiment 70

The compound of embodiment 69, wherein at least one modifiedinternucleoside linkage is a phosphorothioate internucleoside linkage.

Embodiment 71

The compound of embodiment 70, wherein each modified internucleosidelinkage is a phosphorothioate internucleoside linkage.

Embodiment 72

The compound of embodiments 66-69, wherein at least one internucleosidelinkage is a phosphodiester internucleoside linkage.

Embodiment 73

The compound of embodiments 66-71 and 72-73, wherein at least oneinternucleoside linkage is a phosphorothioate linkage and at least oneinternucleoside linkage is a phosphodiester linkage.

Embodiment 74

The compound of embodiments 66-73, wherein at least one nucleosidecomprises a modified nucleobase.

Embodiment 75

The compound of embodiment 74, wherein the modified nucleobase is a5-methylcytosine.

Embodiment 76

The compound of embodiments 66-75, wherein at least one nucleoside ofthe modified oligonucleotide comprises a modified sugar.

Embodiment 77

The compound of embodiment 76, wherein the at least one modified sugaris a bicyclic sugar.

Embodiment 78

The compound of embodiment 77, wherein the bicyclic sugar comprises a4′-CH(R)—O-2′ bridge wherein R is, independently, H, C₁-C₁₂ alkyl, or aprotecting group.

Embodiment 79

The compound of embodiment 78, wherein R is methyl.

Embodiment 80

The compound of embodiment 78, wherein R is H.

Embodiment 81

The compound of embodiment 76, wherein the at least one modified sugarcomprises a 2′-O-methoxyethyl group.

Antisense Compounds

Oligomeric compounds include, but are not limited to, oligonucleotides,oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics,antisense compounds, antisense oligonucleotides, modifiedoligonucleotides, and siRNAs. An oligomeric compound may be “antisense”to a target nucleic acid, meaning that is capable of undergoinghybridization to a target nucleic acid through hydrogen bonding.

In certain embodiments, an antisense compound has a nucleobase sequencethat, when written in the 5′ to 3′ direction, comprises the reversecomplement of the target segment of a target nucleic acid to which it istargeted. In certain such embodiments, an oligonucleotide has anucleobase sequence that, when written in the 5′ to 3′ direction,comprises the reverse complement of the target segment of a targetnucleic acid to which it is targeted.

In certain embodiments, an antisense compound targeted to a SOD-1nucleic acid is 12 to 30 subunits in length. In certain embodiments, anantisense compound targeted to a SOD-1 nucleic acid is 12 to 25 subunitsin length. In certain embodiments, an antisense compound targeted to aSOD-1 nucleic acid is 12 to 22 subunits in length. In certainembodiments, an antisense compound targeted to a SOD-1 nucleic acid is14 to 20 subunits in length. In certain embodiments, an antisensecompound targeted to a SOD-1 nucleic acid is 15 to 25 subunits inlength. In certain embodiments, an antisense compound targeted to aSOD-1 nucleic acid is 18 to 22 subunits in length. In certainembodiments, an antisense compound targeted to a SOD-1 nucleic acid is19 to 21 subunits in length. In certain embodiments, the antisensecompound is 8 to 80, 12 to 50, 13 to 30, 13 to 50, 14 to 30, 14 to 50,15 to 30, 15 to 50, 16 to 30, 16 to 50, 17 to 30, 17 to 50, 18 to 30, 18to 50, 19 to 30, 19 to 50, or 20 to 30 linked subunits in length.

In certain embodiments, an antisense compound targeted to a SOD-1nucleic acid is 12 subunits in length. In certain embodiments, anantisense compound targeted to a SOD-1 nucleic acid is 13 subunits inlength. In certain embodiments, an antisense compound targeted to aSOD-1 nucleic acid is 14 subunits in length. In certain embodiments, anantisense compound targeted to a SOD-1 nucleic acid is 15 subunits inlength. In certain embodiments, an antisense compound targeted to aSOD-1 nucleic acid is 16 subunits in length. In certain embodiments, anantisense compound targeted to a SOD-1 nucleic acid is 17 subunits inlength. In certain embodiments, an antisense compound targeted to aSOD-1 nucleic acid is 18 subunits in length. In certain embodiments, anantisense compound targeted to a SOD-1 nucleic acid is 19 subunits inlength. In certain embodiments, an antisense compound targeted to aSOD-1 nucleic acid is 20 subunits in length. In certain embodiments, anantisense compound targeted to a SOD-1 nucleic acid is 21 subunits inlength. In certain embodiments, an antisense compound targeted to aSOD-1 nucleic acid is 22 subunits in length. In certain embodiments, anantisense compound targeted to a SOD-1 nucleic acid is 23 subunits inlength. In certain embodiments, an antisense compound targeted to aSOD-1 nucleic acid is 24 subunits in length. In certain embodiments, anantisense compound targeted to a SOD-1 nucleic acid is 25 subunits inlength. In certain embodiments, an antisense compound targeted to aSOD-1 nucleic acid is 26 subunits in length. In certain embodiments, anantisense compound targeted to a SOD-1 nucleic acid is 27 subunits inlength. In certain embodiments, an antisense compound targeted to aSOD-1 nucleic acid is 28 subunits in length. In certain embodiments, anantisense compound targeted to a SOD-1 nucleic acid is 29 subunits inlength. In certain embodiments, an antisense compound targeted to aSOD-1 nucleic acid is 30 subunits in length. In certain embodiments, theantisense compound targeted to a SOD-1 nucleic acid is 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 linked subunits in length, ora range defined by any two of the above values. In certain embodimentsthe antisense compound is a modified oligonucleotide, and the linkedsubunits are nucleosides.

In certain embodiments, oligonucleotides targeted to a SOD-1 nucleicacid may be shortened or truncated. For example, a single subunit may bedeleted from the 5′ end (5′ truncation), or alternatively from the 3′end (3′ truncation). A shortened or truncated antisense compoundtargeted to a SOD-1 nucleic acid may have two subunits deleted from the5′ end, or alternatively may have two subunits deleted from the 3′ end,of the antisense compound. Alternatively, the deleted nucleosides may bedispersed throughout the antisense compound, for example, in anantisense compound having one nucleoside deleted from the 5′ end and onenucleoside deleted from the 3′ end.

When a single additional subunit is present in a lengthened antisensecompound, the additional subunit may be located at the 5′ or 3′ end ofthe antisense compound. When two or more additional subunits arepresent, the added subunits may be adjacent to each other, for example,in an antisense compound having two subunits added to the 5′ end (5′addition), or alternatively to the 3′ end (3′ addition), of theantisense compound. Alternatively, the added subunits may be dispersedthroughout the antisense compound, for example, in an antisense compoundhaving one subunit added to the 5′ end and one subunit added to the 3′end.

It is possible to increase or decrease the length of an antisensecompound, such as a modified oligonucleotide, and/or introduce mismatchbases without eliminating activity. For example, in Woolf et al. (Proc.Natl. Acad. Sci. USA 89:7305-7309, 1992), a series of oligonucleotides13-25 nucleobases in length were tested for their ability to inducecleavage of a target RNA in an oocyte injection model. Oligonucleotides25 nucleobases in length with 8 or 11 mismatch bases near the ends ofthe oligonucleotides were able to direct specific cleavage of the targetmRNA, albeit to a lesser extent than oligonucleotides that contained nomismatches. Similarly, target specific cleavage was achieved using 13nucleobase oligonucleotides, including those with 1 or 3 mismatches.

Gautschi et al (J. Natl. Cancer Inst. 93:463-471, March 2001)demonstrated the ability of an oligonucleotide having 100%complementarity to the bcl-2 mRNA and having 3 mismatches to the bcl-xLmRNA to reduce the expression of both bcl-2 and bcl-xL in vitro and invivo. Furthermore, this oligonucleotide demonstrated potent anti-tumoractivity in vivo.

Maher and Dolnick (Nuc. Acid. Res. 16:3341-3358, 1988) tested a seriesof tandem 14 nucleobase oligonucleotides, and a 28 and 42 nucleobaseoligonucleotides comprised of the sequence of two or three of the tandemoligonucleotides, respectively, for their ability to arrest translationof human DHFR in a rabbit reticulocyte assay. Each of the three 14nucleobase oligonucleotides alone was able to inhibit translation,albeit at a more modest level than the 28 or 42 nucleobaseoligonucleotides.

Antisense Compound Motifs

In certain embodiments, antisense compounds targeted to a SOD-1 nucleicacid have chemically modified subunits arranged in patterns, or motifs,to confer to the antisense compounds properties such as enhancedinhibitory activity, increased binding affinity for a target nucleicacid, or resistance to degradation by in vivo nucleases.

Chimeric antisense compounds typically contain at least one regionmodified so as to confer increased resistance to nuclease degradation,increased cellular uptake, increased binding affinity for the targetnucleic acid, and/or increased inhibitory activity. A second region of achimeric antisense compound may optionally serve as a substrate for thecellular endonuclease RNase H, which cleaves the RNA strand of anRNA:DNA duplex.

Antisense compounds having a gapmer motif are considered chimericantisense compounds. In a gapmer an internal region having a pluralityof nucleotides that supports RNaseH cleavage is positioned betweenexternal regions having a plurality of nucleotides that are chemicallydistinct from the nucleosides of the internal region. In the case of anoligonucleotide having a gapmer motif, the gap segment generally servesas the substrate for endonuclease cleavage, while the wing segmentscomprise modified nucleosides. In certain embodiments, the regions of agapmer are differentiated by the types of sugar moieties comprising eachdistinct region. The types of sugar moieties that are used todifferentiate the regions of a gapmer may in some embodiments includeβ-D-ribonucleosides, β-D-deoxyribonucleosides, 2′-modified nucleosides(such 2′-modified nucleosides may include 2′-MOE, and 2′-O—CH₃, amongothers), and bicyclic sugar modified nucleosides (such bicyclic sugarmodified nucleosides may include those having a 4′-(CH₂)n-O-2′ bridge,where n=1 or n=2 and 4′-CH₂—O—CH₂-2′). In certain embodiments, wings mayinclude several modified sugar moieties, including, for example 2′-MOE.In certain embodiments, wings may include several modified andunmodified sugar moieties. In certain embodiments, wings may includevarious combinations of 2′-MOE nucleosides and 2′-deoxynucleosides.

Each distinct region may comprise uniform sugar moieties, variant, oralternating sugar moieties. The wing-gap-wing motif is frequentlydescribed as “X—Y—Z”, where “X” represents the length of the 5′ wing,“Y” represents the length of the gap, and “Z” represents the length ofthe 3′ wing. “X” and “Z” may comprise uniform, variant, or alternatingsugar moieties. In certain embodiments, “X” and “Y” may include one ormore 2′-deoxynucleosides. “Y” may comprise 2′-deoxynucleosides. As usedherein, a gapmer described as “X—Y—Z” has a configuration such that thegap is positioned immediately adjacent to each of the 5′ wing and the 3′wing. Thus, no intervening nucleotides exist between the 5′ wing andgap, or the gap and the 3′ wing. Any of the antisense compoundsdescribed herein can have a gapmer motif. In certain embodiments, “X”and “Z” are the same; in other embodiments they are different.

In certain embodiments, gapmers provided herein include, for example20-mers having a motif of 5-10-5.

In certain embodiments, gapmers provided herein include, for example19-mers having a motif of 5-9-5.

In certain embodiments, gapmers provided herein include, for example18-mers having a motif of 5-8-5.

In certain embodiments, gapmers provided herein include, for example18-mers having a motif of 4-8-5.

In certain embodiments, gapmers provided herein include, for example18-mers having a motif of 5-8-7.

In certain embodiments, gapmers provided herein include, for example18-mers having a motif of 6-8-6.

In certain embodiments, gapmers provided herein include, for example18-mers having a motif of 6-8-5.

In certain embodiments, the modified oligonucleotide contains at leastone 2′-O-methoxyethyl modified nucleoside, at least one cEt modifiednucleoside, and at least one 2′-deoxynucleoside. In certain embodiments,the modified oligonucleotide has a sugar chemistry motif of any of thefollowing:

ekddddddddekekee kekeddddddddekek eeeedddddddddkkee eeeeddddddddekekeeeeeddddddddkekee eeeeddddddddkkeee eeeeeddddddddkkee eeeekddddddddkeeeeeeekdddddddkeeee eeekddddddddkeeee eeekkdddddddkkeee eekkdddddddddkkeeeekkddddddddeeeee eekkddddddddkkeee ekekddddddddeeeee ekekddddddddkekeekekeddddddddeeeee, wherein e = a 2′-O-methoxyethylribose modified sugar,k = a cEt modified sugar, d = a 2′-deoxyribose sugar,Target Nucleic Acids, Target Regions and Nucleotide Sequences

Nucleotide sequences that encode SOD-1 include, without limitation, thefollowing: GENBANK Accession No. NM_000454.4 (incorporated herein as SEQID NO: 1), GENBANK Accession No. NT_011512.10 truncated from nucleotides18693000 to Ser. No. 18/704,000 (incorporated herein as SEQ ID NO: 2),and the complement of GENBANK Accession No. NW_001114168.1 truncatedfrom nucleotides 2258000 to U.S. Pat. No. 2,271,000 (incorporated hereinas SEQ ID NO: 3).

It is understood that the sequence set forth in each SEQ ID NO in theExamples contained herein is independent of any modification to a sugarmoiety, an internucleoside linkage, or a nucleobase. As such, antisensecompounds defined by a SEQ ID NO may comprise, independently, one ormore modifications to a sugar moiety, an internucleoside linkage, or anucleobase. Antisense compounds described by Isis Number (Isis No)indicate a combination of nucleobase sequence and motif.

In certain embodiments, a target region is a structurally defined regionof the target nucleic acid. For example, a target region may encompass a3′ UTR, a 5′ UTR, an exon, an intron, an exon/intron junction, a codingregion, a translation initiation region, translation termination region,or other defined nucleic acid region. The structurally defined regionsfor SOD-1 can be obtained by accession number from sequence databasessuch as NCBI and such information is incorporated herein by reference.In certain embodiments, a target region may encompass the sequence froma 5′ target site of one target segment within the target region to a 3′target site of another target segment within the same target region.

Targeting includes determination of at least one target segment to whichan antisense compound hybridizes, such that a desired effect occurs. Incertain embodiments, the desired effect is a reduction in mRNA targetnucleic acid levels. In certain embodiments, the desired effect isreduction of levels of protein encoded by the target nucleic acid or aphenotypic change associated with the target nucleic acid.

A target region may contain one or more target segments. Multiple targetsegments within a target region may be overlapping. Alternatively, theymay be non-overlapping. In certain embodiments, target segments within atarget region are separated by no more than about 300 nucleotides. Incertain embodiments, target segments within a target region areseparated by a number of nucleotides that is, is about, is no more than,is no more than about, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30,20, or 10 nucleotides on the target nucleic acid, or is a range definedby any two of the preceeding values. In certain embodiments, targetsegments within a target region are separated by no more than, or nomore than about, 5 nucleotides on the target nucleic acid. In certainembodiments, target segments are contiguous. Contemplated are targetregions defined by a range having a starting nucleic acid that is any ofthe 5′ target sites or 3′ target sites listed herein.

Suitable target segments may be found within a 5′ UTR, a coding region,a 3′ UTR, an intron, an exon, or an exon/intron junction. Targetsegments containing a start codon or a stop codon are also suitabletarget segments. A suitable target segment may specifically exclude acertain structurally defined region such as the start codon or stopcodon.

The determination of suitable target segments may include a comparisonof the sequence of a target nucleic acid to other sequences throughoutthe genome. For example, the BLAST algorithm may be used to identifyregions of similarity amongst different nucleic acids. This comparisoncan prevent the selection of antisense compound sequences that mayhybridize in a non-specific manner to sequences other than a selectedtarget nucleic acid (i.e., non-target or off-target sequences).

There may be variation in activity (e.g., as defined by percentreduction of target nucleic acid levels) of the antisense compoundswithin an active target region. In certain embodiments, reductions inSOD-1 mRNA levels are indicative of inhibition of SOD-1 expression.Reductions in levels of a SOD-1 protein are also indicative ofinhibition of target mRNA expression. Phenotypic changes are indicativeof inhibition of SOD-1 expression. Improvement in neurological functionis indicative of inhibition of SOD-1 expression. Improved motor functionis indicative of inhibition of SOD-1 expression.

Hybridization

In some embodiments, hybridization occurs between an antisense compounddisclosed herein and a SOD-1 nucleic acid. The most common mechanism ofhybridization involves hydrogen bonding (e.g., Watson-Crick, Hoogsteenor reversed Hoogsteen hydrogen bonding) between complementarynucleobases of the nucleic acid molecules.

Hybridization can occur under varying conditions. Stringent conditionsare sequence-dependent and are determined by the nature and compositionof the nucleic acid molecules to be hybridized.

Methods of determining whether a sequence is specifically hybridizableto a target nucleic acid are well known in the art. In certainembodiments, the antisense compounds provided herein are specificallyhybridizable with a SOD-1 nucleic acid.

Complementarity

An antisense compound and a target nucleic acid are complementary toeach other when a sufficient number of nucleobases of the antisensecompound can hydrogen bond with the corresponding nucleobases of thetarget nucleic acid, such that a desired effect will occur (e.g.,antisense inhibition of a target nucleic acid, such as a SOD-1 nucleicacid).

Non-complementary nucleobases between an antisense compound and a SOD-1nucleic acid may be tolerated provided that the antisense compoundremains able to specifically hybridize to a target nucleic acid.Moreover, an antisense compound may hybridize over one or more segmentsof a SOD-1 nucleic acid such that intervening or adjacent segments arenot involved in the hybridization event (e.g., a loop structure,mismatch or hairpin structure).

In certain embodiments, the antisense compounds provided herein, or aspecified portion thereof, are, or are at least, 70%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%complementary to a SOD-1 nucleic acid, a target region, target segment,or specified portion thereof. Percent complementarity of an antisensecompound with a target nucleic acid can be determined using routinemethods.

For example, an antisense compound in which 18 of 20 nucleobases of theantisense compound are complementary to a target region, and wouldtherefore specifically hybridize, would represent 90 percentcomplementarity. In this example, the remaining noncomplementarynucleobases may be clustered or interspersed with complementarynucleobases and need not be contiguous to each other or to complementarynucleobases. As such, an antisense compound which is 18 nucleobases inlength having 4 (four) noncomplementary nucleobases which are flanked bytwo regions of complete complementarity with the target nucleic acidwould have 77.8% overall complementarity with the target nucleic acidand would thus fall within the scope of the present invention. Percentcomplementarity of an antisense compound with a region of a targetnucleic acid can be determined routinely using BLAST programs (basiclocal alignment search tools) and PowerBLAST programs known in the art(Altschul et al., J. Mol. Biol., 1990, 215, 403 410; Zhang and Madden,Genome Res., 1997, 7, 649 656). Percent homology, sequence identity orcomplementarity, can be determined by, for example, the Gap program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, Madison Wis.), using defaultsettings, which uses the algorithm of Smith and Waterman (Adv. Appl.Math., 1981, 2, 482 489).

In certain embodiments, the antisense compounds provided herein, orspecified portions thereof, are fully complementary (i.e., 100%complementary) to a target nucleic acid, or specified portion thereof.For example, an antisense compound may be fully complementary to a SOD-1nucleic acid, or a target region, or a target segment or target sequencethereof. As used herein, “fully complementary” means each nucleobase ofan antisense compound is capable of precise base pairing with thecorresponding nucleobases of a target nucleic acid. For example, a 20nucleobase antisense compound is fully complementary to a targetsequence that is 400 nucleobases long, so long as there is acorresponding 20 nucleobase portion of the target nucleic acid that isfully complementary to the antisense compound. Fully complementary canalso be used in reference to a specified portion of the first and/or thesecond nucleic acid. For example, a 20 nucleobase portion of a 30nucleobase antisense compound can be “fully complementary” to a targetsequence that is 400 nucleobases long. The 20 nucleobase portion of the30 nucleobase oligonucleotide is fully complementary to the targetsequence if the target sequence has a corresponding 20 nucleobaseportion wherein each nucleobase is complementary to the 20 nucleobaseportion of the antisense compound. At the same time, the entire 30nucleobase antisense compound may or may not be fully complementary tothe target sequence, depending on whether the remaining 10 nucleobasesof the antisense compound are also complementary to the target sequence.

The location of a non-complementary nucleobase may be at the 5′ end or3′ end of the antisense compound. Alternatively, the non-complementarynucleobase or nucleobases may be at an internal position of theantisense compound. When two or more non-complementary nucleobases arepresent, they may be contiguous (i.e., linked) or non-contiguous. In oneembodiment, a non-complementary nucleobase is located in the wingsegment of a gapmer oligonucleotide.

In certain embodiments, antisense compounds that are, or are up to 11,12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleobases in length comprise nomore than 4, no more than 3, no more than 2, or no more than 1non-complementary nucleobase(s) relative to a target nucleic acid, suchas a SOD-1 nucleic acid, or specified portion thereof.

In certain embodiments, antisense compounds that are, or are up to 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,or 30 nucleobases in length comprise no more than 6, no more than 5, nomore than 4, no more than 3, no more than 2, or no more than 1non-complementary nucleobase(s) relative to a target nucleic acid, suchas a SOD-1 nucleic acid, or specified portion thereof.

The antisense compounds provided herein also include those which arecomplementary to a portion of a target nucleic acid. As used herein,“portion” refers to a defined number of contiguous (i.e. linked)nucleobases within a region or segment of a target nucleic acid. A“portion” can also refer to a defined number of contiguous nucleobasesof an antisense compound. In certain embodiments, the antisensecompounds, are complementary to at least an 8 nucleobase portion of atarget segment. In certain embodiments, the antisense compounds arecomplementary to at least a 9 nucleobase portion of a target segment. Incertain embodiments, the antisense compounds are complementary to atleast a 10 nucleobase portion of a target segment. In certainembodiments, the antisense compounds, are complementary to at least an11 nucleobase portion of a target segment. In certain embodiments, theantisense compounds, are complementary to at least a 12 nucleobaseportion of a target segment. In certain embodiments, the antisensecompounds, are complementary to at least a 13 nucleobase portion of atarget segment. In certain embodiments, the antisense compounds, arecomplementary to at least a 14 nucleobase portion of a target segment.In certain embodiments, the antisense compounds, are complementary to atleast a 15 nucleobase portion of a target segment. Also contemplated areantisense compounds that are complementary to at least a 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, or more nucleobase portion of a targetsegment, or a range defined by any two of these values.

Identity

The antisense compounds provided herein may also have a defined percentidentity to a particular nucleotide sequence, SEQ ID NO, or compoundrepresented by a specific Isis number, or portion thereof. As usedherein, an antisense compound is identical to the sequence disclosedherein if it has the same nucleobase pairing ability. For example, a RNAwhich contains uracil in place of thymidine in a disclosed DNA sequencewould be considered identical to the DNA sequence since both uracil andthymidine pair with adenine. Shortened and lengthened versions of theantisense compounds described herein as well as compounds havingnon-identical bases relative to the antisense compounds provided hereinalso are contemplated. The non-identical bases may be adjacent to eachother or dispersed throughout the antisense compound. Percent identityof an antisense compound is calculated according to the number of basesthat have identical base pairing relative to the sequence to which it isbeing compared.

In certain embodiments, the antisense compounds, or portions thereof,are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%identical to one or more of the antisense compounds or SEQ ID NOs, or aportion thereof, disclosed herein.

In certain embodiments, a portion of the antisense compound is comparedto an equal length portion of the target nucleic acid. In certainembodiments, an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, or 25 nucleobase portion is compared to an equal lengthportion of the target nucleic acid.

In certain embodiments, a portion of the oligonucleotide is compared toan equal length portion of the target nucleic acid. In certainembodiments, an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, or 25 nucleobase portion is compared to an equal lengthportion of the target nucleic acid.

Modifications

A nucleoside is a base-sugar combination. The nucleobase (also known asbase) portion of the nucleoside is normally a heterocyclic base moiety.Nucleotides are nucleosides that further include a phosphate groupcovalently linked to the sugar portion of the nucleoside. For thosenucleosides that include a pentofuranosyl sugar, the phosphate group canbe linked to the 2′, 3′ or 5′ hydroxyl moiety of the sugar.Oligonucleotides are formed through the covalent linkage of adjacentnucleosides to one another, to form a linear polymeric oligonucleotide.Within the oligonucleotide structure, the phosphate groups are commonlyreferred to as forming the internucleoside linkages of theoligonucleotide.

Modifications to antisense compounds encompass substitutions or changesto internucleoside linkages, sugar moieties, or nucleobases. Modifiedantisense compounds are often preferred over native forms because ofdesirable properties such as, for example, enhanced cellular uptake,enhanced affinity for nucleic acid target, increased stability in thepresence of nucleases, or increased inhibitory activity.

Chemically modified nucleosides may also be employed to increase thebinding affinity of a shortened or truncated oligonucleotide for itstarget nucleic acid. Consequently, comparable results can often beobtained with shorter antisense compounds that have such chemicallymodified nucleosides.

Modified Internucleoside Linkages

The naturally occurring internucleoside linkage of RNA and DNA is a 3′to 5′ phosphodiester linkage. Antisense compounds having one or moremodified, i.e. non-naturally occurring, internucleoside linkages areoften selected over antisense compounds having naturally occurringinternucleoside linkages because of desirable properties such as, forexample, enhanced cellular uptake, enhanced affinity for target nucleicacids, and increased stability in the presence of nucleases.

Oligonucleotides having modified internucleoside linkages includeinternucleoside linkages that retain a phosphorus atom as well asinternucleoside linkages that do not have a phosphorus atom.Representative phosphorus containing internucleoside linkages include,but are not limited to, phosphodiesters, phosphotriesters,methylphosphonates, phosphoramidate, and phosphorothioates. Methods ofpreparation of phosphorous-containing and non-phosphorous-containinglinkages are well known.

In certain embodiments, modified oligonucleotides targeted to a SOD-1nucleic acid comprise one or more modified internucleoside linkages. Incertain embodiments, the modified internucleoside linkages areinterspersed throughout the antisense compound. In certain embodiments,the modified internucleoside linkages are phosphorothioate linkages. Incertain embodiments, each internucleoside linkage of a modifiedoligonucleotide is a phosphorothioate internucleoside linkage.

In certain embodiments, the modified oligonucleotides targeted to aSOD-1 nucleic acid comprise one or more phosphodiester internucleosidelinkages. In certain embodiments, modified oligonucleotides targeted toa SOD-1 nucleic acid comprise at least one phosphorothioateinternucleoside linkage and at least one phosphodiester internucleosidelinkage. In certain embodiments, the modified oligonucleotide has amixed backbone motif of the following:

sossssssssoooss, sooossssssssoss, sooosssssssssoss, soosssssssssooss,sooossssssssooss, sooosssssssssooss, sooossssssssssooss,sooosssssssssssooos, soooossssssssssooss, sooosssssssssssooss,sososssssssssssosos, and sooossssssssssoooss, wherein s = aphosphorothioate internucleoside linkage, and o = a phosphodiesterinternucleoside linkage.Modified Sugar Moieties

Antisense compounds of the invention can optionally contain one or morenucleosides wherein the sugar group has been modified. Such sugarmodified nucleosides may impart enhanced nuclease stability, increasedbinding affinity, or some other beneficial biological property to theantisense compounds. In certain embodiments, nucleosides comprisechemically modified ribofuranose ring moieties. Examples of chemicallymodified ribofuranose rings include without limitation, addition ofsubstitutent groups (including 5′ and 2′ substituent groups, bridging ofnon-geminal ring atoms to form bicyclic nucleic acids (BNA), replacementof the ribosyl ring oxygen atom with S, N(R), or C(R₁)(R₂) (R, R₁ and R₂are each independently H, C₁-C₁₂ alkyl or a protecting group) andcombinations thereof. Examples of chemically modified sugars include2′-F-5′-methyl substituted nucleoside (see PCT International ApplicationWO 2008/101157 Published on Aug. 21, 2008 for other disclosed 5′,2′-bissubstituted nucleosides) or replacement of the ribosyl ring oxygen atomwith S with further substitution at the 2′-position (see published U.S.Patent Application US2005-0130923, published on Jun. 16, 2005) oralternatively 5′-substitution of a BNA (see PCT InternationalApplication WO 2007/134181 Published on Nov. 22, 2007 wherein LNA issubstituted with for example a 5′-methyl or a 5′-vinyl group).

Examples of nucleosides having modified sugar moieties include withoutlimitation nucleosides comprising 5′-vinyl, 5′-methyl (R or S), 4′-S,2′-F, 2′-OCH₃, 2′-OCH₂CH₃, 2′-OCH₂CH₂F and 2′-O(CH₂)₂OCH₃ substituentgroups. The substituent at the 2′ position can also be selected fromallyl, amino, azido, thio, O-allyl, O—C₁-C₁₀ alkyl, OCF₃, OCH₂F,O(CH₂)₂SCH₃, O(CH₂)₂—O—N(R_(m))(R_(n)), O—CH₂—C(═O)—N(R_(m))(R_(n)), andO—CH₂—C(═O)—N(R_(l))—(CH₂)₂—N(R_(m))(R_(n)), where each R_(l), R_(m) andR_(n) is, independently, H or substituted or unsubstituted C₁-C₁₀ alkyl.

As used herein, “bicyclic nucleosides” refer to modified nucleosidescomprising a bicyclic sugar moiety. Examples of bicyclic nucleic acids(BNAs) include without limitation nucleosides comprising a bridgebetween the 4′ and the 2′ ribosyl ring atoms. In certain embodiments,antisense compounds provided herein include one or more BNA nucleosideswherein the bridge comprises one of the formulas: 4′-(CH₂)—O-2′ (LNA);4′-(CH₂)—S-2; 4′-(CH₂)₂—O-2′ (ENA); 4′-CH(CH₃)—O-2′ and4′-CH(CH₂OCH₃)—O-2′ (and analogs thereof see U.S. Pat. No. 7,399,845,issued on Jul. 15, 2008); 4′-C(CH₃)(CH₃)—O-2′ (and analogs thereof seePCT/US2008/068922 published as WO/2009/006478, published Jan. 8, 2009);4′-CH₂—N(OCH₃)-2′ (and analogs thereof see PCT/US2008/064591 publishedas WO/2008/150729, published Dec. 11, 2008); 4′-CH₂—O—N(CH₃)-2′ (seepublished U.S. Patent Application US2004-0171570, published Sep. 2,2004); 4′-CH₂—N(R)—O-2′, wherein R is H, C₁-C₁₂ alkyl, or a protectinggroup (see U.S. Pat. No. 7,427,672, issued on Sep. 23, 2008);4′-CH₂—C(H)(CH₃)-2′ (see Chattopadhyaya et al., J. Org. Chem., 2009, 74,118-134); and 4′-CH₂—C(═CH₂)-2′ (and analogs thereof seePCT/US2008/066154 published as WO 2008/154401, published on Dec. 8,2008).

Further bicyclic nucleosides have been reported in published literature(see for example: Srivastava et al., J. Am. Chem. Soc., 2007, 129(26)8362-8379; Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372;Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braaschet al., Chem. Biol., 2001, 8, 1-7; Orum et al., Curr. Opinion Mol.Ther., 2001, 3, 239-243; Wahlestedt et al., Proc. Natl. Acad. Sci.U.S.A, 2000, 97, 5633-5638; Singh et al., Chem. Commun., 1998, 4,455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Kumar et al.,Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al., J. Org.Chem., 1998, 63, 10035-10039; U.S. Pat. Nos. 7,399,845; 7,053,207;7,034,133; 6,794,499; 6,770,748; 6,670,461; 6,525,191; 6,268,490; U.S.Patent Publication Nos.: US2008-0039618; US2007-0287831; US2004-0171570;U.S. patent application Ser. Nos. 12/129,154; 61/099,844; 61/097,787;61/086,231; 61/056,564; 61/026,998; 61/026,995; 60/989,574;International applications WO 2007/134181; WO 2005/021570; WO2004/106356; WO 94/14226; and PCT International Applications Nos.:PCT/US2008/068922; PCT/US2008/066154; and PCT/US2008/064591). Each ofthe foregoing bicyclic nucleosides can be prepared having one or morestereochemical sugar configurations including for exampleα-L-ribofuranose and β-D-ribofuranose (see PCT international applicationPCT/DK98/00393, published on Mar. 25, 1999 as WO 99/14226).

As used herein, “monocylic nucleosides” refer to nucleosides comprisingmodified sugar moieties that are not bicyclic sugar moieties. In certainembodiments, the sugar moiety, or sugar moiety analogue, of a nucleosidemay be modified or substituted at any position.

As used herein, “4′-2′ bicyclic nucleoside” or “4′ to 2′ bicyclicnucleoside” refers to a bicyclic nucleoside comprising a furanose ringcomprising a bridge connecting two carbon atoms of the furanose ringconnects the 2′ carbon atom and the 4′ carbon atom of the sugar ring.

In certain embodiments, bicyclic sugar moieties of BNA nucleosidesinclude, but are not limited to, compounds having at least one bridgebetween the 4′ and the 2′ carbon atoms of the pentofuranosyl sugarmoiety including without limitation, bridges comprising 1 or from 1 to 4linked groups independently selected from —[C(R_(a))(R_(b))]_(n)—,—C(R_(a))═C(R_(b))—, —C(R_(a))═N—, —C(═NR_(a))—, —C(═O)—, —C(═S)—, —O—,—Si(R_(a))₂—, —S(═O)_(x)—, and —N(R_(a))—; wherein: x is 0, 1, or 2; nis 1, 2, 3, or 4; each R_(a) and R_(b) is, independently, H, aprotecting group, hydroxyl, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl,C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substitutedC₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, heterocycleradical, substituted heterocycle radical, heteroaryl, substitutedheteroaryl, C₅-C₇ alicyclic radical, substituted C₅-C₇ alicyclicradical, halogen, OJ₁, NJ₁J₂, SJ₁, N₃, COOJ₁, acyl (C(═O)—H),substituted acyl, CN, sulfonyl (S(═O)₂-J₁), or sulfoxyl (S(═O)-J₁); and

each J₁ and J₂ is, independently, H, C₁-C₁₂ alkyl, substituted C₁-C₁₂alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl,substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, acyl(C(═O)—H), substituted acyl, a heterocycle radical, a substitutedheterocycle radical, C₁-C₁₂ aminoalkyl, substituted C₁-C₁₂ aminoalkyl ora protecting group.

In certain embodiments, the bridge of a bicyclic sugar moiety is,—[C(R_(a))(R_(b))]_(n)—, —[C(R_(a))(R_(b))]_(n)—O—,—C(R_(a)R_(b))—N(R)—O— or —C(R_(a)R_(b))—O—N(R)—. In certainembodiments, the bridge is 4′-CH₂—2′, 4′-(CH₂)₂-2′, 4′-(CH₂)₃-2′,4′-CH₂—O-2′, 4′-(CH₂)₂—O-2′, 4′-CH₂—O—N(R)-2′ and 4′-CH₂—N(R)—O-2′-wherein each R is, independently, H, a protecting group or C₁-C₁₂ alkyl.

In certain embodiments, bicyclic nucleosides are further defined byisomeric configuration. For example, a nucleoside comprising a4′-(CH₂)—O-2′ bridge, may be in the α-L configuration or in the β-Dconfiguration. Previously, α-L-methyleneoxy (4′-CH₂—O-2′) BNA's havebeen incorporated into antisense oligonucleotides that showed antisenseactivity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).

In certain embodiments, bicyclic nucleosides include those having a 4′to 2′ bridge wherein such bridges include without limitation,α-L-4′-(CH₂)—O-2′, β-D-4′-CH₂—O-2′, 4′-(CH₂)₂—O-2′,4′-CH₂—O—N(R)-2′4′-CH₂—N(R)—O-2′, 4′-CH(CH₃)—O-2′, 4′-CH₂—S-2′,4′-CH₂—N(R)-2′, 4′-CH₂—CH(CH₃)-2′, and 4′-(CH₂)₃-2′, wherein R is H, aprotecting group or C₁-C₁₂ alkyl.

In certain embodiment, bicyclic nucleosides have the formula:

wherein:

Bx is a heterocyclic base moiety;

-Q_(a)-Q_(b)-Q_(c)- is —CH₂—N(R_(c))—CH₂—, —C(═O)—N(R_(c))—CH₂—,—CH₂—O—N(R_(c))—, —CH₂—N(R_(c))—O— or —N(R_(c))—O—CH₂;

R_(c) is C₁-C₁₂ alkyl or an amino protecting group; and

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium.

In certain embodiments, bicyclic nucleosides have the formula:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium;

Z_(a) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₁-C₆alkyl, substituted C₂-C₆ alkenyl, substituted C₂-C₆ alkynyl, acyl,substituted acyl, substituted amide, thiol or substituted thiol.

In one embodiment, each of the substituted groups, is, independently,mono or poly substituted with substituent groups independently selectedfrom halogen, oxo, hydroxyl, OJ_(c), SJ_(c), N₃, OC(═X)J_(c), andNJ_(e)C(═X)NJ_(c)J_(d), wherein each J_(c), J_(d) and J_(e) is,independently, H, C₁-C₆ alkyl, or substituted C₁-C₆ alkyl and X is O orNJ_(c).

In certain embodiments, bicyclic nucleosides have the formula:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium;

Z_(b) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₁-C₆alkyl, substituted C₂-C₆ alkenyl, substituted C₂-C₆ alkynyl orsubstituted acyl (C(═O)—).

In certain embodiments, bicyclic nucleosides have the formula:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium;

R_(d) is C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl,substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

each q_(a), q_(b), q_(c) and q_(d) is, independently, H, halogen, C₁-C₆alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl, C₁-C₆ alkoxyl,substituted C₁-C₆ alkoxyl, acyl, substituted acyl, C₁-C₆ aminoalkyl orsubstituted C₁-C₆ aminoalkyl;

In certain embodiments, bicyclic nucleosides have the formula:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium;

q_(a), q_(b), q_(e) and q_(f) are each, independently, hydrogen,halogen, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl,substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl,C₁-C₁₂ alkoxy, substituted C₁-C₁₂ alkoxy, OJ_(j), SJ_(j), SOJ_(j),SO₂J_(j), NJ_(j)J_(k), N₃, CN, C(═O)OJ_(j), C(═O)NJ_(j)J_(k),C(═O)J_(j), O—C(═O)NJ_(j)J_(k), N(H)C(═NH)NJ_(j)J_(k),N(H)C(═O)NJ_(j)J_(k) or N(H)C(═S)NJ_(j)J_(k);

or q_(e) and q_(f) together are ═C(q_(g))(q_(h));

q_(g) and q_(h) are each, independently, H, halogen, C₁-C₁₂ alkyl orsubstituted C₁-C₁₂ alkyl.

The synthesis and preparation of adenine, cytosine, guanine,5-methyl-cytosine, thymine and uracil bicyclic nucleosides having a4′-CH₂—O-2′ bridge, along with their oligomerization, and nucleic acidrecognition properties have been described (Koshkin et al., Tetrahedron,1998, 54, 3607-3630). The synthesis of bicyclic nucleosides has alsobeen described in WO 98/39352 and WO 99/14226.

Analogs of various bicyclic nucleosides that have 4′ to 2′ bridginggroups such as 4′-CH₂—O-2′ and 4′-CH₂—S-2′, have also been prepared(Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222).Preparation of oligodeoxyribonucleotide duplexes comprising bicyclicnucleosides for use as substrates for nucleic acid polymerases has alsobeen described (Wengel et al., WO 99/14226). Furthermore, synthesis of2′-amino-BNA, a novel conformationally restricted high-affinityoligonucleotide analog has been described in the art (Singh et al., J.Org. Chem., 1998, 63, 10035-10039). In addition, 2′-amino- and2′-methylamino-BNA's have been prepared and the thermal stability oftheir duplexes with complementary RNA and DNA strands has beenpreviously reported.

In certain embodiments, bicyclic nucleosides have the formula:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium;

each q_(i), q_(j), q_(k) and q_(l) is, independently, H, halogen, C₁-C₁₂alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₁-C₁₂ alkoxyl,substituted C₁-C₁₂ alkoxyl, OJ_(j), SJ_(j), SOJ_(j), SO₂J_(j),NJ_(j)J_(k), N₃, CN, C(═O)OJ_(j), C(═O)NJ_(j)J_(k), C(═O)J_(j),O—C(═O)NJ_(j)J_(k), N(H)C(═NH)NJ_(j)J_(k), N(H)C(═O)NJ_(j)J_(k) orN(H)C(═S)NJ_(j)J_(k); and

q_(i) and q_(j) or q_(l) and q_(k) together are ═C(q_(g))(q_(h)),wherein q_(g) and q_(h) are each, independently, H, halogen, C₁-C₁₂alkyl or substituted C₁-C₁₂ alkyl.

One carbocyclic bicyclic nucleoside having a 4′-(CH₂)₃-2′ bridge and thealkenyl analog bridge 4′-CH═CH—CH₂-2′ have been described (Frier et al.,Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al., J.Org. Chem., 2006, 71, 7731-7740). The synthesis and preparation ofcarbocyclic bicyclic nucleosides along with their oligomerization andbiochemical studies have also been described (Srivastava et al., J. Am.Chem. Soc. 2007, 129(26), 8362-8379).

In certain embodiments, bicyclic nucleosides include, but are notlimited to, (A) α-L-methyleneoxy (4′-CH₂—O-2′) BNA, (B) β-D-methyleneoxy(4′-CH₂—O-2′) BNA, (C) ethyleneoxy (4′-(CH₂)₂—O-2′) BNA, (D) aminooxy(4′-CH₂—O—N(R)-2′) BNA, (E) oxyamino (4′-CH₂—N(R)—O-2′) BNA, (F)methyl(methyleneoxy) (4′-CH(CH₃)—O-2′) BNA (also referred to asconstrained ethyl or cEt), (G) methylene-thio (4′-CH₂—S-2′) BNA, (H)methylene-amino (4′-CH₂—N(R)-2′) BNA, (I) methyl carbocyclic(4′-CH₂—CH(CH₃)-2′) BNA, (J) propylene carbocyclic (4′-(CH₂)₃-2′) BNA,and (K) vinyl BNA as depicted below.

wherein Bx is the base moiety and R is, independently, H, a protectinggroup, C₁-C₆ alkyl or C₁-C₆ alkoxy.

As used herein, the term “modified tetrahydropyran nucleoside” or“modified THP nucleoside” means a nucleoside having a six-memberedtetrahydropyran “sugar” substituted for the pentofuranosyl residue innormal nucleosides and can be referred to as a sugar surrogate. ModifiedTHP nucleosides include, but are not limited to, what is referred to inthe art as hexitol nucleic acid (HNA), anitol nucleic acid (ANA),manitol nucleic acid (MNA) (see Leumann, Bioorg. Med. Chem., 2002, 10,841-854) or fluoro HNA (F-HNA) having a tetrahydropyranyl ring system asillustrated below.

In certain embodiment, sugar surrogates are selected having the formula:

wherein:

Bx is a heterocyclic base moiety;

T₃ and T₄ are each, independently, an internucleoside linking grouplinking the tetrahydropyran nucleoside analog to the oligomeric compoundor one of T₃ and T₄ is an internucleoside linking group linking thetetrahydropyran nucleoside analog to an oligomeric compound oroligonucleotide and the other of T₃ and T₄ is H, a hydroxyl protectinggroup, a linked conjugate group or a 5′ or 3′-terminal group;

q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each independently, H, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆alkynyl or substituted C₂-C₆ alkynyl; and

one of R₁ and R₂ is hydrogen and the other is selected from halogen,substituted or unsubstituted alkoxy, NJ₁J₂, SJ₁, N₃, OC(═X)J₁,OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂ and CN, wherein X is O, S or NJ₁ and each J₁,J₂ and J₃ is, independently, H or C₁-C₆ alkyl.

In certain embodiments, q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each H. Incertain embodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ isother than H. In certain embodiments, at least one of q₁, q₂, q₃, q₄,q₅, q₆ and q₇ is methyl. In certain embodiments, THP nucleosides areprovided wherein one of R₁ and R₂ is F. In certain embodiments, R₁ isfluoro and R₂ is H; R₁ is methoxy and R₂ is H, and R₁ is methoxyethoxyand R₂ is H.

In certain embodiments, sugar surrogates comprise rings having more than5 atoms and more than one heteroatom. For example nucleosides comprisingmorpholino sugar moieties and their use in oligomeric compounds has beenreported (see for example: Braasch et al., Biochemistry, 2002, 41,4503-4510; and U.S. Pat. Nos. 5,698,685; 5,166,315; 5,185,444; and5,034,506). As used here, the term “morpholino” means a sugar surrogatehaving the following formula:

In certain embodiments, morpholinos may be modified, for example byadding or altering various substituent groups from the above morpholinostructure. Such sugar surrogates are referred to herein as “modifiedmorpholinos.”

Combinations of modifications are also provided without limitation, suchas 2′-F-5′-methyl substituted nucleosides (see PCT InternationalApplication WO 2008/101157 published on Aug. 21, 2008 for otherdisclosed 5′, 2′-bis substituted nucleosides) and replacement of theribosyl ring oxygen atom with S and further substitution at the2′-position (see published U.S. Patent Application US2005-0130923,published on Jun. 16, 2005) or alternatively 5′-substitution of abicyclic nucleic acid (see PCT International Application WO 2007/134181,published on Nov. 22, 2007 wherein a 4′-CH₂—O-2′ bicyclic nucleoside isfurther substituted at the 5′ position with a 5′-methyl or a 5′-vinylgroup). The synthesis and preparation of carbocyclic bicyclicnucleosides along with their oligomerization and biochemical studieshave also been described (see, e.g., Srivastava et al., J. Am. Chem.Soc. 2007, 129(26), 8362-8379).

In certain embodiments, antisense compounds comprise one or moremodified cyclohexenyl nucleosides, which is a nucleoside having asix-membered cyclohexenyl in place of the pentofuranosyl residue innaturally occurring nucleosides. Modified cyclohexenyl nucleosidesinclude, but are not limited to those described in the art (see forexample commonly owned, published PCT Application WO 2010/036696,published on Apr. 10, 2010, Robeyns et al., J. Am. Chem. Soc., 2008,130(6), 1979-1984; Horvath et al., Tetrahedron Letters, 2007, 48,3621-3623; Nauwelaerts et al., J. Am. Chem. Soc., 2007, 129(30),9340-9348; Gu et al., Nucleosides, Nucleotides & Nucleic Acids, 2005,24(5-7), 993-998; Nauwelaerts et al., Nucleic Acids Research, 2005,33(8), 2452-2463; Robeyns et al., Acta Crystallographica, Section F:Structural Biology and Crystallization Communications, 2005, F61(6),585-586; Gu et al., Tetrahedron, 2004, 60(9), 2111-2123; Gu et al.,Oligonucleotides, 2003, 13(6), 479-489; Wang et al., J. Org. Chem.,2003, 68, 4499-4505; Verbeure et al., Nucleic Acids Research, 2001,29(24), 4941-4947; Wang et al., J. Org. Chem., 2001, 66, 8478-82; Wanget al., Nucleosides, Nucleotides & Nucleic Acids, 2001, 20(4-7),785-788; Wang et al., J. Am. Chem., 2000, 122, 8595-8602; Published PCTapplication, WO 06/047842; and Published PCT Application WO 01/049687;the text of each is incorporated by reference herein, in theirentirety). Certain modified cyclohexenyl nucleosides have Formula X.

wherein independently for each of said at least one cyclohexenylnucleoside analog of Formula X:

Bx is a heterocyclic base moiety;

T₃ and T₄ are each, independently, an internucleoside linking grouplinking the cyclohexenyl nucleoside analog to an antisense compound orone of T₃ and T₄ is an internucleoside linking group linking thetetrahydropyran nucleoside analog to an antisense compound and the otherof T₃ and T₄ is H, a hydroxyl protecting group, a linked conjugategroup, or a 5′- or 3′-terminal group; and

q₁, q₂, q₃, q₄, q₅, q₆, q₇, q₈ and q₉ are each, independently, H, C₁-C₆alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl or other sugarsubstituent group.

Many other monocyclic, bicyclic and tricyclic ring systems are known inthe art and are suitable as sugar surrogates that can be used to modifynucleosides for incorporation into oligomeric compounds as providedherein (see for example review article: Leumann, Christian J. Bioorg. &Med. Chem., 2002, 10, 841-854). Such ring systems can undergo variousadditional substitutions to further enhance their activity.

As used herein, “2′-modified sugar” means a furanosyl sugar modified atthe 2′ position. In certain embodiments, such modifications includesubstituents selected from: a halide, including, but not limited tosubstituted and unsubstituted alkoxy, substituted and unsubstitutedthioalkyl, substituted and unsubstituted amino alkyl, substituted andunsubstituted alkyl, substituted and unsubstituted allyl, andsubstituted and unsubstituted alkynyl. In certain embodiments, 2′modifications are selected from substituents including, but not limitedto: O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)F,O(CH₂)_(n)ONH₂, OCH₂C(═O)N(H)CH₃, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃]₂, wheren and m are from 1 to about 10. Other 2′-substituent groups can also beselected from: C₁-C₁₂ alkyl, substituted alkyl, alkenyl, alkynyl,alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, F,CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving pharmacokinetic properties, or a group for improving thepharmacodynamic properties of an antisense compound, and othersubstituents having similar properties. In certain embodiments, modifiednucleosides comprise a 2′-MOE side chain (Baker et al., J. Biol. Chem.,1997, 272, 11944-12000). Such 2′-MOE substitution have been described ashaving improved binding affinity compared to unmodified nucleosides andto other modified nucleosides, such as 2′-O-methyl, O-propyl, andO-aminopropyl. Oligonucleotides having the 2′-MOE substituent also havebeen shown to be antisense inhibitors of gene expression with promisingfeatures for in vivo use (Martin, Helv. Chim. Acta, 1995, 78, 486-504;Altmann et al., Chimia, 1996, 50, 168-176; Altmann et al., Biochem. Soc.Trans., 1996, 24, 630-637; and Altmann et al., Nucleosides Nucleotides,1997, 16, 917-926).

As used herein, “2′-modified” or “2′-substituted” refers to a nucleosidecomprising a sugar comprising a substituent at the 2′ position otherthan H or OH. 2′-modified nucleosides, include, but are not limited to,bicyclic nucleosides wherein the bridge connecting two carbon atoms ofthe sugar ring connects the 2′ carbon and another carbon of the sugarring; and nucleosides with non-bridging 2′substituents, such as allyl,amino, azido, thio, O-allyl, O—C₁-C₁₀ alkyl, —OCF₃, O—(CH₂)₂—O—CH₃,2′-O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(R_(m))(R_(n)), orO—CH₂—C(═O)—N(R_(m))(R_(n)), where each R_(m) and R_(n) is,independently, H or substituted or unsubstituted C₁-C₁₀ alkyl.2′-modified nucleosides may further comprise other modifications, forexample at other positions of the sugar and/or at the nucleobase.

As used herein, “2′-F” refers to a nucleoside comprising a sugarcomprising a fluoro group at the 2′ position of the sugar ring.

As used herein, “2′-OMe” or “2′-OCH₃”, “2′-O-methyl” or “2′-methoxy”each refers to a nucleoside comprising a sugar comprising an —OCH₃ groupat the 2′ position of the sugar ring.

As used herein, “MOE” or “2′-MOE” or “2′-OCH₂CH₂OCH₃” or“2′-O-methoxyethyl” each refers to a nucleoside comprising a sugarcomprising a —OCH₂CH₂OCH₃ group at the 2′ position of the sugar ring.

Methods for the preparations of modified sugars are well known to thoseskilled in the art. Some representative U.S. patents that teach thepreparation of such modified sugars include without limitation, U.S.:4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137;5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722;5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,670,633;5,700,920; 5,792,847 and 6,600,032 and International ApplicationPCT/US2005/019219, filed Jun. 2, 2005 and published as WO 2005/121371 onDec. 22, 2005, and each of which is herein incorporated by reference inits entirety.

As used herein, “oligonucleotide” refers to a compound comprising aplurality of linked nucleosides. In certain embodiments, one or more ofthe plurality of nucleosides is modified. In certain embodiments, anoligonucleotide comprises one or more ribonucleosides (RNA) and/ordeoxyribonucleosides (DNA).

In nucleotides having modified sugar moieties, the nucleobase moieties(natural, modified or a combination thereof) are maintained forhybridization with an appropriate nucleic acid target.

In certain embodiments, antisense compounds comprise one or morenucleosides having modified sugar moieties. In certain embodiments, themodified sugar moiety is 2′-MOE. In certain embodiments, the 2′-MOEmodified nucleosides are arranged in a gapmer motif. In certainembodiments, the modified sugar moiety is a bicyclic nucleoside having a(4′-CH(CH₃)—O-2′) bridging group. In certain embodiments, the(4′-CH(CH₃)—O-2′) modified nucleosides are arranged throughout the wingsof a gapmer motif.

Compositions and Methods for Formulating Pharmaceutical Compositions

Oligonucleotides may be admixed with pharmaceutically acceptable activeor inert substances for the preparation of pharmaceutical compositionsor formulations. Compositions and methods for the formulation ofpharmaceutical compositions are dependent upon a number of criteria,including, but not limited to, route of administration, extent ofdisease, or dose to be administered.

An antisense compound targeted to a SOD-1 nucleic acid can be utilizedin pharmaceutical compositions by combining the antisense compound witha suitable pharmaceutically acceptable diluent or carrier. Apharmaceutically acceptable diluent includes phosphate-buffered saline(PBS). PBS is a diluent suitable for use in compositions to be deliveredparenterally. Accordingly, in one embodiment, employed in the methodsdescribed herein is a pharmaceutical composition comprising an antisensecompound targeted to a SOD-1 nucleic acid and a pharmaceuticallyacceptable diluent. In certain embodiments, the pharmaceuticallyacceptable diluent is PBS. In certain embodiments, the antisensecompound is a modified oligonucleotide.

Pharmaceutical compositions comprising antisense compounds encompass anypharmaceutically acceptable salts, esters, or salts of such esters, orany other oligonucleotide which, upon administration to an animal,including a human, is capable of providing (directly or indirectly) thebiologically active metabolite or residue thereof. Accordingly, forexample, the disclosure is also drawn to pharmaceutically acceptablesalts of antisense compounds, prodrugs, pharmaceutically acceptablesalts of such prodrugs, and other bioequivalents. Suitablepharmaceutically acceptable salts include, but are not limited to,sodium and potassium salts.

A prodrug can include the incorporation of additional nucleosides at oneor both ends of an antisense compound which are cleaved by endogenousnucleases within the body, to form the active antisense compound.

Conjugated Antisense Compounds

Antisense compounds may be covalently linked to one or more moieties orconjugates which enhance the activity, cellular distribution or cellularuptake of the resulting oligonucleotides. Typical conjugate groupsinclude cholesterol moieties and lipid moieties. Additional conjugategroups include carbohydrates, phospholipids, biotin, phenazine, folate,phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines,coumarins, and dyes.

Antisense compounds can also be modified to have one or more stabilizinggroups that are generally attached to one or both termini of antisensecompounds to enhance properties such as, for example, nucleasestability. Included in stabilizing groups are cap structures. Theseterminal modifications protect the antisense compound having terminalnucleic acid from exonuclease degradation, and can help in deliveryand/or localization within a cell. The cap can be present at the5′-terminus (5′-cap), or at the 3′-terminus (3′-cap), or can be presenton both termini. Cap structures are well known in the art and include,for example, inverted deoxy abasic caps. Further 3′ and 5′-stabilizinggroups that can be used to cap one or both ends of an antisense compoundto impart nuclease stability include those disclosed in WO 03/004602published on Jan. 16, 2003.

Cell Culture and Antisense Compounds Treatment

The effects of antisense compounds on the level, activity or expressionof SOD-1 nucleic acids can be tested in vitro in a variety of celltypes. Cell types used for such analyses are available from commericalvendors (e.g. American Type Culture Collection, Manassas, Va.; Zen-Bio,Inc., Research Triangle Park, N.C.; Clonetics Corporation, Walkersville,Md.) and are cultured according to the vendor's instructions usingcommercially available reagents (e.g. Invitrogen Life Technologies,Carlsbad, Calif.). Illustrative cell types include, but are not limitedto, HepG2 cells, Hep3B cells, primary hepatocytes, A431 cells, andSH-SY5Y cells.

In Vitro Testing of Oligonucleotides

Described herein are methods for treatment of cells witholigonucleotides, which can be modified appropriately for treatment withother antisense compounds.

Cells may be treated with oligonucleotides when the cells reachapproximately 60-80% confluency in culture.

One reagent commonly used to introduce oligonucleotides into culturedcells includes the cationic lipid transfection reagent LIPOFECTIN(Invitrogen, Carlsbad, Calif.). Oligonucleotides may be mixed withLIPOFECTIN in OPTI-MEM 1 (Invitrogen, Carlsbad, Calif.) to achieve thedesired final concentration of oligonucleotide and a LIPOFECTINconcentration that may range from 2 to 12 ug/mL per 100 nMoligonucleotide.

Another reagent used to introduce oligonucleotides into cultured cellsincludes LIPOFECTAMINE (Invitrogen, Carlsbad, Calif.). Oligonucleotideis mixed with LIPOFECTAMINE in OPTI-MEM 1 reduced serum medium(Invitrogen, Carlsbad, Calif.) to achieve the desired concentration ofoligonucleotide and a LIPOFECTAMINE concentration that may range from 2to 12 ug/mL per 100 nM oligonucleotide.

Another technique used to introduce oligonucleotides into cultured cellsincludes electroporation.

Cells are treated with oligonucleotides by routine methods. Cells may beharvested 16-24 hours after oligonucleotide treatment, at which time RNAor protein levels of target nucleic acids are measured by methods knownin the art and described herein. In general, when treatments areperformed in multiple replicates, the data are presented as the averageof the replicate treatments.

The concentration of oligonucleotide used varies from cell line to cellline. Methods to determine the optimal oligonucleotide concentration fora particular cell line are well known in the art. Oligonucleotides aretypically used at concentrations ranging from 1 nM to 300 nM whentransfected with LIPOFECTAMINE. Oligonucleotides are used at higherconcentrations ranging from 625 to 20,000 nM when transfected usingelectroporation.

RNA Isolation

RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA.Methods of RNA isolation are well known in the art. RNA is preparedusing methods well known in the art, for example, using the TRIZOLReagent (Invitrogen, Carlsbad, Calif.) according to the manufacturer'srecommended protocols.

Analysis of Inhibition of Target Levels or Expression

Inhibition of levels or expression of a SOD-1 nucleic acid can beassayed in a variety of ways known in the art. For example, targetnucleic acid levels can be quantitated by, e.g., Northern blot analysis,competitive polymerase chain reaction (PCR), or quantitative real-timePCR. RNA analysis can be performed on total cellular RNA or poly(A)+mRNA. Methods of RNA isolation are well known in the art. Northern blotanalysis is also routine in the art. Quantitative real-time PCR can beconveniently accomplished using the commercially available ABI PRISM7600, 7700, or 7900 Sequence Detection System, available from PE-AppliedBiosystems, Foster City, Calif. and used according to manufacturer'sinstructions.

Quantitative Real-Time PCR Analysis of Target RNA Levels

Quantitation of target RNA levels may be accomplished by quantitativereal-time PCR using the ABI PRISM 7600, 7700, or 7900 Sequence DetectionSystem (PE-Applied Biosystems, Foster City, Calif.) according tomanufacturer's instructions. Methods of quantitative real-time PCR arewell known in the art.

Prior to real-time PCR, the isolated RNA is subjected to a reversetranscriptase (RT) reaction, which produces complementary DNA (cDNA)that is then used as the substrate for the real-time PCR amplification.The RT and real-time PCR reactions are performed sequentially in thesame sample well. RT and real-time PCR reagents may be obtained fromInvitrogen (Carlsbad, Calif.). RT real-time-PCR reactions are carriedout by methods well known to those skilled in the art.

Gene (or RNA) target quantities obtained by real time PCR are normalizedusing either the expression level of a gene whose expression isconstant, such as cyclophilin A, or by quantifying total RNA usingRIBOGREEN (Invitrogen, Inc. Carlsbad, Calif.). Cyclophilin A expressionis quantified by real time PCR, by being run simultaneously with thetarget, multiplexing, or separately. Total RNA is quantified usingRIBOGREEN RNA quantification reagent (Invetrogen, Inc. Eugene, Oreg.).Methods of RNA quantification by RIBOGREEN are SOD-1ght in Jones, L. J.,et al, (Analytical Biochemistry, 1998, 265, 368-374). A CYTOFLUOR 4000instrument (PE Applied Biosystems) is used to measure RIBOGREENfluorescence.

Probes and primers are designed to hybridize to a SOD-1 nucleic acid.Methods for designing real-time PCR probes and primers are well known inthe art, and may include the use of software such as PRIMER EXPRESSSoftware (Applied Biosystems, Foster City, Calif.).

Analysis of Protein Levels

Antisense inhibition of SOD-1 nucleic acids can be assessed by measuringSOD-1 protein levels. Protein levels of SOD-1 can be evaluated orquantitated in a variety of ways well known in the art, such asimmunoprecipitation, Western blot analysis (immunoblotting),enzyme-linked immunosorbent assay (ELISA), quantitative protein assays,protein activity assays (for example, caspase activity assays),immunohistochemistry, immunocytochemistry or fluorescence-activated cellsorting (FACS). Antibodies directed to a target can be identified andobtained from a variety of sources, such as the MSRS catalog ofantibodies (Aerie Corporation, Birmingham, Mich.), or can be preparedvia conventional monoclonal or polyclonal antibody generation methodswell known in the art. In certain embodiments, the compounds hereinprovide improved reduction in protein levels.

In Vivo Testing of Antisense Compounds

Antisense compounds, for example, modified oligonucleotides, are testedin animals to assess their ability to inhibit expression of SOD-1 andproduce phenotypic changes, such as, improved motor function. In certainembodiments, motor function is measured by walking initiation analysis,rotarod, grip strength, pole climb, open field performance, balancebeam, hindpaw footprint testing in the animal. Testing may be performedin normal animals, or in experimental disease models. For administrationto animals, oligonucleotides are formulated in a pharmaceuticallyacceptable diluent, such as phosphate-buffered saline. Administrationincludes parenteral routes of administration, such as intraperitoneal,intravenous, and subcutaneous. Oligonucleotide dosage and dosingfrequency depends upon multiple factors such as, but not limited to,route of administration and animal body weight. Following a period oftreatment with oligonucleotides, RNA is isolated from CNS tissue or CSFand changes in SOD-1 nucleic acid expression are measured.

Certain Indications

In certain embodiments, provided herein are methods, compounds, andcompositions of treating an individual comprising administering one ormore pharmaceutical compositions described herein. In certainembodiments, the individual has a neurodegenerative disease. In certainembodiments, the individual is at risk for developing aneurodegenerative disease, including, but not limited to, amyotrophiclateral sclerosis (ALS). In certain embodiments, the individual has beenidentified as having a SOD-1 associated disease. In certain embodiments,provided herein are methods for prophylactically reducing SOD-1expression in an individual. Certain embodiments include treating anindividual in need thereof by administering to an individual atherapeutically effective amount of an antisense compound targeted to aSOD-1 nucleic acid.

In one embodiment, administration of a therapeutically effective amountof an antisense compound targeted to a SOD-1 nucleic acid is accompaniedby monitoring of SOD-1 levels in an individual, to determine anindividual's response to administration of the antisense compound. Anindividual's response to administration of the antisense compound may beused by a physician to determine the amount and duration of therapeuticintervention.

In certain embodiments, administration of an antisense compound targetedto a SOD-1 nucleic acid results in reduction of SOD-1 expression by atleast 15, 20, 25, 30, 35, 40, 45, 50, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99, or 100%, or a range defined by any two of these values. In certainembodiments, administration of an antisense compound targeted to a SOD-1nucleic acid results in improved motor function in an animal. In certainembodiments, administration of a SOD-1 antisense compound improves motorfunction by at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, or 100%, or a range defined by any two of these values.

In certain embodiments, pharmaceutical compositions comprising anantisense compound targeted to SOD-1 are used for the preparation of amedicament for treating a patient suffering or susceptible to aneurodegenerative disease including amyotrophic lateral sclerosis (ALS).

Certain Comparator Compositions

Antisense oligonucleotides targeting human SOD-1 were described in anearlier publication (see WO 2005/040180, incorporated by referenceherein, in its entirety). Several oligonucleotides (ISIS 333611, ISIS146144, ISIS 146145, ISIS 150437, ISIS 150441, ISIS 150443, ISIS 150444,ISIS 150445, ISIS 150446, ISIS 150447, ISIS 150448, ISIS 150449, ISIS150452, ISIS 150454, ISIS 150458, ISIS 150460, ISIS 150462-150467, ISIS150470, ISIS 150472, ISIS 150474, ISIS 150475, ISIS 150476, ISIS150479-150483, ISIS 150488, ISIS 150489, ISIS 150490, ISIS150491-150493, ISIS 150495-150498, ISIS 150511, ISIS 333605, ISIS333606, ISIS 333609-333617, ISIS 333619, ISIS 333620-333636, ISIS333638, and ISIS 333640) described therein, were used as comparatorcompounds throughout select screens for new antisense compoundsdescribed herein.

In certain embodiments, ISIS 333611, a 5-10-5 MOE gapmer, having asequence of (from 5′ to 3′) CCGTCGCCCTTCAGCACGCA (incorporated herein asSEQ ID NO: 21), wherein each internucleoside linkage is aphosphorothioate linkage, each cytosine is a 5-methylcytosine, and eachof nucleosides 1-5 and 16-20 (from 5′ to 3′) comprise a2′-O-methoxyethyl moiety was used as a comparator compound. ISIS 333611was selected as a comparator compound because it exhibited high levelsof dose-dependent inhibition in various studies as described in WO2005/040180. Additionally, phase 1 human clinical trials were completedusing ISIS 333611. See, MILLER et al., “An antisense oligonucleotideagainst SOD1 delivered intrathecally for patients with SOD1 familialamyotrophic lateral sclerosis: a phase 1, randomised, first-in-manstudy” Lancet Neurol. (2013) 12(5): 435-442. Thus, ISIS 333611 wasdeemed a highly efficacious and potent compound with an acceptablesafety profile (such that it was tested in human patients).

In certain embodiments, the compounds described herein benefit from oneor more improved properties relative to the antisense compoundsdescribed in WO 2005/040180. Some of the improved properties aredemonstrated in the examples provided herein. In certain embodiments,compounds described herein are more efficacious, potent, and/ortolerable in various in vitro and in vivo studies than comparatorcompounds described herein, including ISIS 333611. In certainembodiments, ISIS 666853, ISIS 666859, ISIS 666919, ISIS 666921, ISIS666922, ISIS 666869, ISIS 666870, and ISIS 666867 are more efficaciousand/or potent in various in vitro and in vivo studies than comparatorcompounds described herein, including ISIS 333611. In certainembodiments, ISIS 666853, ISIS 666859, ISIS 666919, ISIS 666921, ISIS666922, ISIS 666869, ISIS 666870, and ISIS 666867 are more tolerable inone or more tolerability assays in animals than comparator compoundsdescribed herein, including ISIS 333611. This is despite 333611 beingsufficiently well tolerated to progress to human clinical trials.

In certain embodiments, certain compounds described herein are moreefficacious than comparator compounds by virtue of an in vitro IC50 ofless than 2 μM, less than 1.9 μM, less than 1.8 μM, less than 1.7 μM,less than 1.6 μM, less than 1.5 μM, less than 1.4 μM, less than 1.3 μM,less than 1.2 μM, less than 1.1 μM, less than 1 μM, less than 0.9 μM,less than 0.8 μM, less than 0.7 μM, less than 0.6 μM, or less than 0.5μM less than 0.4 μM, less than 0.3 μM, less than 0.2 μM, less than 0.1μM, when tested in human cells, for example, in the HepG2 A431 orSH-SY5Y cell lines (For example, see Examples 6-11).

In certain embodiments, certain compounds described herein are moreefficacious than comparator compounds by virtue of their ability toinhibit SOD-1 expression in vivo. In certain embodiments, the compoundsinhibit SOD-1 in lumbar spinal cord and cervical spinal cord by at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90% or at least 95% in, for example, a transgenic animalmodel.

In certain embodiments, certain compounds described herein are moretolerable than comparator compounds on the basis of reduced microglialmarker levels (e.g., IBA1), reduced astrocytic marker levels (e.g.,GFAP), and/or FOB scores in rats, mice, and/or monkeys. See, forexample, Examples 14, 15, 18, and 19.

ISIS 666853

For example, as provided in Example 12 (hereinbelow), ISIS 666853achieved 81% inhibition in lumbar spinal cord and 74% in cervical spinalcord of an SOD-1 transgenic rat model when dosed with 30 μL of 16.67mg/ml solution of oligonucleotide diluted in PBS (500 μg final dose),whereas ISIS 333611 achieved 51% inhibition in lumbar spinal cord and47% inhibition in cervical spinal cord.

For example, as provided in Example 14 (hereinbelow), ISIS 666853achieved a FOB score of 0 whereas ISIS 333611 achieved a FOB score of 4in Sprague-Dawley rats after 3 hours when treated with 3 mg ofoligonucleotide. Microglial marker (IBA1) levels and astrocytic marker(GFAP) levels were also reduced in ISIS 666853 treated rats as comparedto ISIS 333611 treated rats.

For example, as provided in Example 15 (hereinbelow), ISIS 666853achieved an ED₅₀ of 81.3 and 242.6 in lumbar tissue and cervical tissue(respectively) in SOD-1 transgenic rats when treated intrathecally with10, 30, 100, 300, or 3000 μg of oligonucleotide. ED₅₀ in lumbar andcervical tissues could not be calculated in ISIS 333611 treatedtransgenic rats because the highest concentration tested (3000 μg) filedto inhibit human SOD-1 mRNA greater than 55-65%.

For example, as provided in Example 16 (hereinbelow), at doses of 1 mgand 3 mg ISIS 666853 achieved 3 hour FOB scores of 0.0 and 0.5(respectively) whereas ISIS 333611 achieved FOB scores of 3.0 and 4.9(respectively). At doses of 1 mg and 3 mg ISIS 666853 achieved 8 weekFOB scores of 0.0 and 0.0 (respectively) whereas ISIS 333611 achievedFOB scores of 0.0 and 1.2 (respectively).

For example, as provided in Example 17 (hereinbelow), ISIS 666853achieved an ED₅₀ of 136 and 188 in lumbar tissue and cortex tissue(respectively) whereas ISIS 333611 achieved an ED₅₀ of 401 and 786 inlumbar tissue and cortex tissue (respectively) in SOD-1 transgenic micewhen treated with an intracerebral ventricular bolus of 10, 30, 100,300, or 700 μg of oligonucleotide.

For example, as provided in Example 18 (hereinbelow), ISIS 666853achieved a FOB score of 1.25 whereas ISIS 333611 achieved a FOB score of6.5 in C57bl6 mice after 3 hours when treated with 700 μg ofoligonucleotide. Microglial marker (IBA1) levels and astrocytic marker(GFAP) levels were also reduced in ISIS 666853 treated mice as comparedto ISIS 333611 treated mice.

ISIS 666859

For example, as provided in Example 12 (hereinbelow), ISIS 666859achieved 79% inhibition in lumbar spinal cord and 64% inhibition incervical spinal cord of an SOD-1 transgenic rat model when dosed with 30μL of 16.67 mg/ml solution of oligonucleotide diluted in PBS (500 μgfinal dose), whereas ISIS 333611 achieved 51% inhibition in lumbarspinal cord and 47% inhibition in cervical spinal cord.

For example, as provided in Example 14 (hereinbelow), ISIS 666859achieved a FOB score of 1 whereas ISIS 333611 achieved a FOB score of 4in Sprague-Dawley rats after 3 hours when treated with 3 mg ofoligonucleotide. Microglial marker (IBA1) levels and astrocytic marker(GFAP) levels were also reduced in ISIS 666859 treated rats as comparedto ISIS 333611 treated rats.

For example, as provided in Example 15 (hereinbelow), ISIS 666859achieved an ED₅₀ of 74.0 and 358.8 in lumbar tissue and cervical tissue(respectively) in SOD-1 transgenic rats when treated intrathecally with10, 30, 100, 300, or 3000 μg of oligonucleotide. ED₅₀ in lumbar andcervical tissues could not be calculated in ISIS 333611 treatedtransgenic rats because the highest concentration tested (3000 μg) filedto inhibit human SOD-1 mRNA greater than 55-65%.

For example, as provided in Example 16 (hereinbelow), at doses of 1 mgand 3 mg ISIS 666859 achieved 3 hour FOB scores of 0.0 and 2.1(respectively) whereas ISIS 333611 achieved FOB scores of 3.0 and 4.9(respectively). At doses of 1 mg and 3 mg ISIS 666859 achieved 8 weekFOB scores of 0.0 and 0.3 (respectively) whereas ISIS 333611 achievedFOB scores of 0.0 and 1.2 (respectively).

For example, as provided in Example 17 (hereinbelow), ISIS 666859achieved an ED₅₀ of 106 and 206 in lumbar tissue and cortex tissue(respectively) whereas ISIS 333611 achieved an ED₅₀ of 401 and 786 inlumbar tissue and cortex tissue (respectively) in SOD-1 transgenic micewhen treated with an intracerebral ventricular bolus of 10, 30, 100,300, or 700 μg of oligonucleotide.

For example, as provided in Example 18 (hereinbelow), ISIS 666859achieved a FOB score of 1.75 whereas ISIS 333611 achieved a FOB score of6.5 in C57bl6 mice after 3 hours when treated with 700 μg ofoligonucleotide. Microglial marker (IBA1) levels and astrocytic marker(GFAP) levels were also reduced in ISIS 666859 treated mice as comparedto ISIS 333611 treated mice.

ISIS 666919

For example, as provided in Example 12 (hereinbelow), ISIS 666919achieved 76% inhibition in lumbar spinal cord and 68% in cervical spinalcord of an SOD-1 transgenic rat model when dosed with 30 μL of 16.67mg/ml solution of oligonucleotide diluted in PBS (500 μg final dose),whereas ISIS 333611 achieved 51% inhibition in lumbar spinal cord and47% inhibition in cervical spinal cord.

For example, as provided in Example 14 (hereinbelow), ISIS 666919achieved a FOB score of 2 whereas ISIS 333611 achieved a FOB score of 4in Sprague-Dawley rats after 3 hours when treated with 3 mg ofoligonucleotide. Microglial marker (IBA1) levels and astrocytic marker(GFAP) levels were also reduced in ISIS 666919 treated rats as comparedto ISIS 333611 treated rats.

For example, as provided in Example 15 (hereinbelow), ISIS 666919achieved an ED₅₀ of 104.1 and 613.5 in lumbar tissue and cervical tissue(respectively) in SOD-1 transgenic rats when treated intrathecally with10, 30, 100, 300, or 3000 μg of oligonucleotide. ED₅₀ in lumbar andcervical tissues could not be calculated in ISIS 333611 treatedtransgenic rats because the highest concentration tested (3000 μg) filedto inhibit human SOD-1 mRNA greater than 55-65%.

For example, as provided in Example 16 (hereinbelow), at doses of 1 mgand 3 mg ISIS 666919 achieved 3 hour FOB scores of 1.3 and 3.5(respectively) whereas ISIS 333611 achieved FOB scores of 3.0 and 4.9(respectively). At doses of 1 mg and 3 mg ISIS 666919 achieved 8 weekFOB scores of 0.0 and 0.1 (respectively) whereas ISIS 333611 achievedFOB scores of 0.0 and 1.2 (respectively).

For example, as provided in Example 17 (hereinbelow), ISIS 666919achieved an ED₅₀ of 168 in lumbar tissue whereas ISIS 333611 achieved anED₅₀ of 401 in lumbar tissue in SOD-1 transgenic mice when treated withan intracerebral ventricular bolus of 10, 30, 100, 300, or 700 ofoligonucleotide.

For example, as provided in Example 18 (hereinbelow), ISIS 666919achieved a FOB score of 0.0 whereas ISIS 333611 achieved a FOB score of6.5 in C57bl6 mice after 3 hours when treated with 700 μg ofoligonucleotide. Microglial marker (IBA1) levels and astrocytic marker(GFAP) levels were also reduced in ISIS 666919 treated mice as comparedto ISIS 333611 treated mice.

ISIS 666921

For example, as provided in Example 12 (hereinbelow), ISIS 66621achieved 71% inhibition in lumbar spinal cord and 65% in cervical spinalcord of an SOD-1 transgenic rat model when dosed with 30 μL of 16.67mg/ml solution of oligonucleotide diluted in PBS (500 μg final dose),whereas ISIS 333611 achieved 51% inhibition in lumbar spinal cord and47% inhibition in cervical spinal cord.

For example, as provided in Example 14 (hereinbelow), ISIS 666921achieved a FOB score of 2 whereas ISIS 333611 achieved a FOB score of 4in Sprague-Dawley rats after 3 hours when treated with 3 mg ofoligonucleotide. Microglial marker (IBA1) levels and astrocytic marker(GFAP) levels were also reduced in ISIS 666919 treated rats as comparedto ISIS 333611 treated rats.

ISIS 666922

For example, as provided in Example 12 (hereinbelow), ISIS 666922achieved 67% inhibition in lumbar spinal cord and 62% in cervical spinalcord of an SOD-1 transgenic rat model when dosed with 30 μL of 16.67mg/ml solution of oligonucleotide diluted in PBS (500 μg final dose),whereas ISIS 333611 achieved 51% inhibition in lumbar spinal cord and47% inhibition in cervical spinal cord.

For example, as provided in Example 14 (hereinbelow), ISIS 666922achieved a FOB score of 3 whereas ISIS 333611 achieved a FOB score of 4in Sprague-Dawley rats after 3 hours when treated with 3 mg ofoligonucleotide. Microglial marker (IBA1) levels and astrocytic marker(GFAP) levels were also reduced in ISIS 666919 treated rats as comparedto ISIS 333611 treated rats.

ISIS 666869

For example, as provided in Example 12 (hereinbelow), ISIS 666869achieved 82% inhibition in lumbar spinal cord and 81% in cervical spinalcord of an SOD-1 transgenic rat model when dosed with 30 μL of 16.67mg/ml solution of oligonucleotide diluted in PBS (500 μg final dose),whereas ISIS 333611 achieved 51% inhibition in lumbar spinal cord and47% inhibition in cervical spinal cord.

ISIS 666870

For example, as provided in Example 12 (hereinbelow), ISIS 666870achieved 76% inhibition in lumbar spinal cord and 68% in cervical spinalcord of an SOD-1 transgenic rat model when dosed with 30 μL of 16.67mg/ml solution of oligonucleotide diluted in PBS (500 μg final dose),whereas ISIS 333611 achieved 51% inhibition in lumbar spinal cord and47% inhibition in cervical spinal cord.

For example, as provided in Example 15 (hereinbelow), ISIS 666870achieved an ED₅₀ of 139.4 and 1111 in lumbar tissue and cervical tissue(respectively) in SOD-1 transgenic rats when treated intrathecally with10, 30, 100, 300, or 3000 μg of oligonucleotide. ED₅₀ in lumbar andcervical tissues could not be calculated in ISIS 333611 treatedtransgenic rats because the highest concentration tested (3000 μg) filedto inhibit human SOD-1 mRNA greater than 55-65%.

For example, as provided in Example 17 (hereinbelow), ISIS 666870achieved an ED₅₀ of 148 and 409 in lumbar tissue and cortex tissue(respectively) whereas ISIS 333611 achieved an ED₅₀ of 401 and 786 inlumbar tissue and cortex tissue (respectively) in SOD-1 transgenic micewhen treated with an intracerebral ventricular bolus of 10, 30, 100,300, or 700 μg of oligonucleotide.

For example, as provided in Example 18 (hereinbelow), ISIS 666870achieved a FOB score of 4.75 whereas ISIS 333611 achieved a FOB score of6.5 in C57bl6 mice after 3 hours when treated with 700 μg ofoligonucleotide.

ISIS 666867

For example, as provided in Example 12 (hereinbelow), ISIS 666867achieved 59% inhibition in lumbar spinal cord and 48% in cervical spinalcord of an SOD-1 transgenic rat model when dosed with 30 μL of 16.67mg/ml solution of oligonucleotide diluted in PBS (500 μg final dose),whereas ISIS 333611 achieved 51% inhibition in lumbar spinal cord and47% inhibition in cervical spinal cord.

Certain Compositions

1. ISIS 666853

In certain embodiments, ISIS 666853 is characterized as a 5-10-5 MOEgapmer, having a sequence of (from 5′ to 3′) CAGGATACATTTCTACAGCT(incorporated herein as SEQ ID NO: 725), wherein each of nucleosides 1-5and 16-20 are 2′-O-methoxyethylribose modified nucleosides, and each ofnucleosides 6-15 are 2′-deoxynucleosides, wherein the internucleosidelinkages between nucleosides 2 to 3, 4 to 5, 16 to 17, and 18 to 19 arephosphodiester linkages and the internucleoside linkages betweennucleosides 1 to 2, 3 to 4, 5 to 6, 6 to 7, 7 to 8, 8 to 9, 9 to 10, 10to 11, 11 to 12, 12 to 13, 13 to 14, 14 to 15, 15 to 16, 17 to 18, and19 to 20 are phosphorothioate linkages, and wherein each cytosine is a5′-methylcytosine.

In certain embodiments, ISIS 666853 is described by the followingchemical notation: mCes Aeo Ges Geo Aes Tds Ads mCds Ads Tds Tds TdsmCds Tds Ads mCeo Aes Geo mCes Te; wherein,

A=an adenine,

mC=a 5′-methylcytosine

G=a guanine,

T=a thymine,

e=a 2′-O-methoxyethylribose modified sugar,

d=a 2′-deoxyribose sugar,

s=a phosphorothioate internucleoside linkage, and

o=a phosphodiester internucleoside linkage.

In certain embodiments, ISIS 666853 is described by the followingchemical structure:

2. ISIS 666859

In certain embodiments, ISIS 666859 is characterized as a modifiedoligonucleotide having the nucleobase sequence (from 5′ to 3′)TTAATGTTTATCAGGAT (incorporated herein as SEQ ID NO: 1351), consistingof seventeen nucleosides, wherein each of nucleosides 1-4 and 15-17 are2′-O-methoxyethylribose nucleosides, wherein each of nucleosides 13 and14 are cEt modified nucleosides, wherein each of nucleosides 5-12 are2′-deoxyribonucleosides, wherein the internucleoside linkages betweennucleosides 2 to 3, 3 to 4, 13 to 14, 14 to 15 are phosphodiesterlinkages and the internucleoside linkages between nucleosides 1 to 2, 4to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, 9 to 10, 10 to 11, 11 to 12, 12 to13, 15 to 16, and 16 to 17 are phosphorothioate linkages, and whereineach cytosine is a 5′-methylcytosine.

In certain embodiments, ISIS 666859 is described by the followingchemical notation: Tes Teo Aeo Aes Tds Gds Tds Tds Tds Ads Tds mCds AkoGko Ges Aes Te; wherein,

A=an adenine,

mC=a 5′-methylcytosine

G=a guanine,

T=a thymine,

e=a 2′-O-methoxyethylribose modified sugar,

k=a cEt modified sugar,

d=a 2′-deoxyribose sugar,

s=a phosphorothioate internucleoside linkage, and

o=a phosphodiester internucleoside linkage.

In certain embodiments, ISIS 666859 is described by the followingchemical structure:

3. ISIS 666919

In certain embodiments, ISIS 666919 is characterized as a modifiedoligonucleotide having the nucleobase sequence (from 5′ to 3′)GGATACATTTCTACAGC (incorporated herein as SEQ ID NO: 1342), consistingof seventeen nucleosides, wherein each of nucleosides 1-4 and 16-17 are2′-O-methoxyethylribose modified nucleosides, wherein each ofnucleosides 14 and 15 are cEt modified nucleosides, wherein each ofnucleosides 5-13 are 2′-deoxyribonucleosides, wherein theinternucleoside linkages between nucleosides 2 to 3, 3 to 4, 4 to 5, and14 to 15 are phosphodiester linkages and the internucleoside linkagesbetween nucleosides 1 to 2, 5 to 6, 6 to 7, 7 to 8, 8 to 9, 9 to 10, 10to 11, 11 to 12, 12 to 13, 13 to 14, 15 to 16, and 16 to 17 arephosphorothioate linkages, and wherein each cytosine is a5′-methylcytosine.

In certain embodiments, ISIS 666919 is described by the followingchemical notation: Ges Geo Aeo Teo Ads mCds Ads Tds Tds Tds mCds Tds AdsmCko Aks Ges mCe; wherein,

A=an adenine,

mC=a 5′-methylcytosine

G=a guanine,

T=a thymine,

e=a 2′-O-methoxyethylribose modified sugar,

k=a cEt modified sugar,

d=a 2′-deoxyribose sugar,

s=a phosphorothioate internucleoside linkage, and

o=a phosphodiester internucleoside linkage.

In certain embodiments, ISIS 666919 is described by the followingchemical structure:

4. ISIS 666921

In certain embodiments, ISIS 666921 is characterized as a modifiedoligonucleotide having the nucleobase sequence (from 5′ to 3′)GGATACATTTCTACAGC (incorporated herein as SEQ ID NO: 1342), consistingof seventeen nucleosides, wherein each of nucleosides 1-5 and 16-17 are2′-O-methoxyethylribose modified nucleosides, wherein each ofnucleosides 14-15 are cEt modified nucleosides, wherein each ofnucleosides 6-13 are 2′-deoxyribonucleosides, wherein theinternucleoside linkages between nucleosides 2 to 3, 3 to 4, 4 to 5, and14 to 15 are phosphodiester linkages and the internucleoside linkagesbetween nucleosides 1 to 2, 5 to 6, 6 to 7, 7 to 8, 8 to 9, 9 to 10, 10to 11, 11 to 12, 12 to 13, 13 to 14, 15 to 16, and 16 to 17 arephosphorothioate linkages, and wherein each cytosine is a5′-methylcytosine.

In certain embodiments, ISIS 666921 is described by the followingchemical notation: Ges Geo Aeo Teo Aes mCds Ads Tds Tds Tds mCds Tds AdsmCko Aks Ges mCe; wherein,

A=an adenine,

mC=a 5′-methylcytosine

G=a guanine,

T=a thymine,

e=a 2′-O-methoxyethylribose modified sugar,

k=a cEt modified sugar,

d=a 2′-deoxyribose sugar,

s=a phosphorothioate internucleoside linkage, and

o=a phosphodiester internucleoside linkage.

In certain embodiments, ISIS 666921 is described by the followingchemical structure:

5. ISIS 666922

In certain embodiments, ISIS 666922 is characterized as a modifiedoligonucleotide having the nucleobase sequence (from 5′ to 3′)GGATACATTTCTACAGC (incorporated herein as SEQ ID NO: 1342), consistingof seventeen nucleosides, wherein each of nucleosides 1-4 and 15-17 are2′-O-methoxyethylribose modified nucleosides, wherein each ofnucleosides 5 and 14 are cEt modified nucleosides, wherein each ofnucleosides 6-13 are 2′-deoxyribonucleosides, wherein theinternucleoside linkages between nucleosides 2 to 3, 3 to 4, 4 to 5, and14 to 15 are phosphodiester linkages and the internucleoside linkagesbetween nucleosides 1 to 2, 5 to 6, 6 to 7, 7 to 8, 8 to 9, 9 to 10, 10to 11, 11 to 12, 12 to 13, 13 to 14, 15 to 16, and 16 to 17 arephosphorothioate linkages, and wherein each cytosine is a5′-methylcytosine.

In certain embodiments, ISIS 666922 is described by the followingchemical notation: Ges Geo Aeo Teo Aks mCds Ads Tds Tds Tds mCds Tds AdsmCko Aes Ges mCe; wherein,

A=an adenine,

mC=a 5′-methylcytosine

G=a guanine,

T=a thymine,

e=a 2′-O-methoxyethylribose modified sugar,

k=a cEt modified sugar,

d=a 2′-deoxyribose sugar,

s=a phosphorothioate internucleoside linkage, and

o=a phosphodiester internucleoside linkage.

In certain embodiments, ISIS 666922 is described by the followingchemical structure:

6. ISIS 666869

In certain embodiments, ISIS 666869 is characterized as a modifiedoligonucleotide having the nucleobase sequence (from 5′ to 3′)AGTGTTTAATGTTTATC (incorporated herein as SEQ ID NO: 1173), consistingof seventeen nucleosides, wherein each of nucleosides 1, 3, 14, and16-17 are 2′-O-methoxyethylribose modified nucleosides, wherein each ofnucleosides 2, 4, 13, and 15 are cEt modified nucleosides, wherein eachof nucleosides 5-12 are 2′-deoxyribonucleosides, wherein theinternucleoside linkages between nucleosides 2 to 3, 3 to 4, 13 to 14,and 14 to 15 are phosphodiester linkages and the internucleosidelinkages between nucleosides 1 to 2, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8to 9, 9 to 10, 10 to 11, 11 to 12, 12 to 13, 15 to 16, and 16 to 17 arephosphorothioate linkages, and wherein each cytosine is a5′-methylcytosine.

In certain embodiments, ISIS 666869 is described by the followingchemical notation: Aes Gko Teo Gks Tds Tds Tds Ads Ads Tds Gds Tds TkoTeo Aks Tes mCe; wherein,

A=an adenine,

mC=a 5′-methylcytosine

G=a guanine,

T=a thymine,

e=a 2′-O-methoxyethylribose modified sugar,

k=a cEt modified sugar,

d=a 2′-deoxyribose sugar,

s=a phosphorothioate internucleoside linkage, and

o=a phosphodiester internucleoside linkage.

In certain embodiments, ISIS 666869 is described by the followingchemical structure:

7. ISIS 666870

In certain embodiments, ISIS 666870 is characterized as a modifiedoligonucleotide having the nucleobase sequence (from 5′ to 3′)AGTGTTTAATGTTTATC (incorporated herein as SEQ ID NO: 1173), consistingof seventeen nucleosides, wherein each of nucleosides 1, 3, 13-17 are2′-O-methoxyethylribose modified nucleosides, wherein each ofnucleosides 2 and 4 are cEt modified nucleosides, wherein each ofnucleosides 5-12 are 2′-deoxyribonucleosides, wherein theinternucleoside linkages between nucleosides 2 to 3, 3 to 4, 13 to 14,and 14 to 15 are phosphodiester linkages and the internucleosidelinkages between nucleosides 1 to 2, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8to 9, 9 to 10, 10 to 11, 11 to 12, 12 to 13, 15 to 16, and 16 to 17 arephosphorothioate linkages, and wherein each cytosine is a5′-methylcytosine.

In certain embodiments, ISIS 666870 is described by the followingchemical notation: Aes Gko Teo Gks Tds Tds Tds Ads Ads Tds Gds Tds TeoTeo Aes Tes mCe; wherein,

A=an adenine,

mC=a 5′-methylcytosine

G=a guanine,

T=a thymine,

e=a 2′-O-methoxyethylribose modified sugar,

k=a cEt modified sugar,

d=a 2′-deoxyribose sugar,

s=a phosphorothioate internucleoside linkage, and

o=a phosphodiester internucleoside linkage.

In certain embodiments, ISIS 666870 is described by the followingchemical structure:

8. ISIS 666867

In certain embodiments, ISIS 666867 is characterized as a modifiedoligonucleotide having the nucleobase sequence (from 5′ to 3′)AGTGTTTAATGTTTATC (incorporated herein as SEQ ID NO: 1173), consistingof seventeen nucleosides, wherein each of nucleosides 1-2 and 13-17 are2′-O-methoxyethylribose modified nucleosides, wherein each ofnucleosides 3 and 4 are cEt modified nucleosides, wherein each ofnucleosides 5-12 are 2′-deoxyribonucleosides, wherein theinternucleoside linkages between nucleosides 2 to 3, 3 to 4, 13 to 14,and 14 to 15 are phosphodiester linkages and the internucleosidelinkages between nucleosides 1 to 2, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8to 9, 9 to 10, 10 to 11, 11 to 12, 12 to 13, 15 to 16, and 16 to 17 arephosphorothioate linkages, and wherein each cytosine is a5′-methylcytosine.

In certain embodiments, ISIS 666867 is described by the followingchemical notation: Aes Geo Tko Gks Tds Tds Tds Ads Ads Tds Gds Tds TeoTeo Aes Tes mCe; wherein,

A=an adenine,

mC=a 5′-methylcytosine

G=a guanine,

T=a thymine,

e=a 2′-O-methoxyethylribose modified sugar,

k=a cEt modified sugar,

d=a 2′-deoxyribose sugar,

s=a phosphorothioate internucleoside linkage, and

o=a phosphodiester internucleoside linkage.

In certain embodiments, ISIS 666867 is described by the followingchemical structure:

EXAMPLES

Non-Limiting Disclosure and Incorporation by Reference

While certain compounds, compositions, and methods described herein havebeen described with specificity in accordance with certain embodiments,the following examples serve only to illustrate the compounds describedherein and are not intended to limit the same. Each of the referencesrecited in the present application is incorporated herein by referencein its entirety.

Example 1: Inhibition of Human Superoxide Dismutase 1, Soluble (SOD-1)in HepG2 Cells by MOE Gapmers

Modified oligonucleotides were designed targeting a superoxide dismutase1, soluble (SOD-1) nucleic acid and were tested for their effects onSOD-1 mRNA in vitro. ISIS 146144, ISIS 146145, ISIS 150437, ISIS 150441,ISIS 150443, ISIS 150444, ISIS 150445, ISIS 150446, ISIS 150447, ISIS150448, ISIS 150449, ISIS 150452, ISIS 150454, ISIS 150458, ISIS 150460,ISIS 150462-150467, ISIS 150470, ISIS 150472, ISIS 150474, ISIS 150475,ISIS 150476, ISIS 150479-150483, ISIS 150488, ISIS 150489, ISIS 150490,ISIS 150491-150493, ISIS 150495-150498, ISIS 150511, ISIS 333605, ISIS333606, ISIS 333609-333617, ISIS 333619, ISIS 333620-333636, ISIS333638, and ISIS 333640, previously disclosed in WO 2005/040180, werealso included in this assay. ISIS 333611, previously disclosed in WO2005/040180, was also designated as a benchmark or comparatoroligonucleotide. ISIS 333611 was recently tested in human clinicaltrials. See, MILLER et al., “An antisense oligonucleotide against SOD1delivered intrathecally for patients with SOD1 familial amyotrophiclateral sclerosis: a phase 1, randomised, first-in-man study” LancetNeurol. (2013) 12(5): 435-442.

The modified oligonucleotides were tested in a series of experimentsthat had similar culture conditions. The results for each experiment arepresented in separate tables shown below. Cultured HepG2 cells at adensity of 20,000 cells per well were transfected using electroporationwith 7,000 nM modified oligonucleotide. After a treatment period ofapproximately 24 hours, RNA was isolated from the cells and SOD-1 mRNAlevels were measured by quantitative real-time PCR.

Human primer probe set RTS3898 (forward sequence CTCTCAGGAGACCATTGCATCA,designated herein as SEQ ID NO: 11; reverse sequenceTCCTGTCTTTGTACTTTCTTCATTTCC; designated herein as SEQ ID NO: 12; probesequence CCGCACACTGGTGGTCCATGAAAA, designated herein as SEQ ID NO: 13)was used to measure mRNA levels. In cases where the oligonucleotideoverlapped the amplicon of the primer probe set, an alternative primerprobe set, HTS90 (forward sequence CGTGGCCTAGCGAGTTATGG, designatedherein as SEQ ID NO: 14; reverse sequence GAAATTGATGATGCCCTGCA;designated herein as SEQ ID NO: 15; probe sequenceACGAAGGCCGTGTGCGTGCTGX, designated herein as SEQ ID NO: 16), was used tomeasure mRNA levels. SOD-1 mRNA levels were adjusted according to totalRNA content, as measured by RIBOGREEN®. Results are presented as percentinhibition of SOD-1, relative to untreated control cells. ‘n.d.’indicates that inhibition levels were not measured using the particularprimer probe set.

The newly designed modified oligonucleotides in the Tables below weredesigned as 5-10-5 MOE gapmers. The 5-10-5 MOE gapmers are 20nucleosides in length, wherein the central gap segment is comprised often 2′-deoxyribonucleosides and is flanked by wing segments on the 5′direction and the 3′ directions comprising five nucleosides each. Eachnucleoside in the 5′ wing segment and each nucleoside in the 3′ wingsegment has a 2′-MOE modification. The internucleoside linkagesthroughout each gapmer are phosphorothioate linkages. All cytosineresidues throughout each gapmer are 5-methylcytosines. “Start site”indicates the 5′-most nucleoside to which the gapmer is targeted in thehuman gene sequence. “Stop site” indicates the 3′-most nucleoside towhich the gapmer is targeted human gene sequence. Each gapmer listed inthe Tables below is targeted to either the human SOD-1 mRNA, designatedherein as SEQ ID NO: 1 (GENBANK Accession No. NM_000454.4) or the humanSOD-1 genomic sequence, designated herein as SEQ ID NO: 2 (GENBANKAccession No. NT_011512.10 truncated from nucleotides 18693000 to Ser.No. 18/704,000). ‘n/a’ indicates that the modified oligonucleotide doesnot target that particular gene sequence with 100% complementarity.

TABLE 1 Percent Inhibition of SOD-1 mRNA by 5-10-5 MOE gapmers targetingSEQ ID NO: 1 and/or 2 SEQ SEQ SEQ SEQ ID ID % ID ID NO: 1 NO: 1inhibition NO: 2 NO: 2 SEQ ISIS Start Stop with Start Stop ID NO SiteSite Sequence RTS3898 Site Site NO 590065 1 20 CGCCCACTCTGGCCCCAAAC 7807 826 118 590066 35 54 CCGCGACTACTTTATAGGCC 5 841 860 119 333611 167186 CCGTCGCCCTTCAGCACGCA 85 973 992 21 590067 202 221CCTTCTGCTCGAAATTGATG 74 1008 1027 120 590068 203 222TCCTTCTGCTCGAAATTGAT 58 n/a n/a 121 590069 204 223 TTCCTTCTGCTCGAAATTGA50 n/a n/a 122 590070 205 224 TTTCCTTCTGCTCGAAATTG 47 n/a n/a 123 590071206 225 CTTTCCTTCTGCTCGAAATT 31 n/a n/a 124 590072 207 226ACTTTCCTTCTGCTCGAAAT 42 n/a n/a 125 590073 208 227 TACTTTCCTTCTGCTCGAAA38 n/a n/a 126 590074 209 228 TTACTTTCCTTCTGCTCGAA 33 n/a n/a 127 590075210 229 ATTACTTTCCTTCTGCTCGA 39 n/a n/a 128 590076 211 230CATTACTTTCCTTCTGCTCG 28 n/a n/a 129 590077 212 231 CCATTACTTTCCTTCTGCTC58 n/a n/a 130 590078 213 232 TCCATTACTTTCCTTCTGCT 58 n/a n/a 131 590079214 233 GTCCATTACTTTCCTTCTGC 69 n/a n/a 132 590080 215 234GGTCCATTACTTTCCTTCTG 68 n/a n/a 133 590081 216 235 TGGTCCATTACTTTCCTTCT61 n/a n/a 134 590082 217 236 CTGGTCCATTACTTTCCTTC 69 n/a n/a 135 590083218 237 ACTGGTCCATTACTTTCCTT 54 4972 4991 136 150445 219 238CACTGGTCCATTACTTTCCT 84 4973 4992 22 590084 220 239 TCACTGGTCCATTACTTTCC65 4974 4993 137 590085 221 240 TTCACTGGTCCATTACTTTC 45 4975 4994 138590086 222 241 CTTCACTGGTCCATTACTTT 43 4976 4995 139 590087 223 242CCTTCACTGGTCCATTACTT 67 4977 4996 140 590088 224 243ACCTTCACTGGTCCATTACT 59 4978 4997 141 436841 225 244CACCTTCACTGGTCCATTAC 65 4979 4998 142 150446 226 245ACACCTTCACTGGTCCATTA 83 4980 4999 23 393336 227 246 CACACCTTCACTGGTCCATT81 4981 5000 143 150447 228 247 CCACACCTTCACTGGTCCAT 89 4982 5001 24590089 229 248 CCCACACCTTCACTGGTCCA 82 4983 5002 144 590090 230 249CCCCACACCTTCACTGGTCC 89 4984 5003 145 590091 231 250TCCCCACACCTTCACTGGTC 84 4985 5004 146 590092 232 251TTCCCCACACCTTCACTGGT 61 4986 5005 147 590093 233 252CTTCCCCACACCTTCACTGG 60 4987 5006 148 590094 234 253GCTTCCCCACACCTTCACTG 78 4988 5007 149 590095 235 254TGCTTCCCCACACCTTCACT 72 4989 5008 150 590096 236 255ATGCTTCCCCACACCTTCAC 76 4990 5009 151 393337 237 256AATGCTTCCCCACACCTTCA 76 4991 5010 152 590097 238 257TAATGCTTCCCCACACCTTC 68 4992 5011 153 590098 264 283TCCATGCAGGCCTTCAGTCA 63 5018 5037 154 590099 265 284ATCCATGCAGGCCTTCAGTC 64 5019 5038 155 590100 266 285AATCCATGCAGGCCTTCAGT 52 5020 5039 156 590101 267 286GAATCCATGCAGGCCTTCAG 53 5021 5040 157 590102 268 287GGAATCCATGCAGGCCTTCA 65 5022 5041 158 393339 269 288TGGAATCCATGCAGGCCTTC 43 5023 5042 159 590103 270 289ATGGAATCCATGCAGGCCTT 56 5024 5043 160 590104 271 290CATGGAATCCATGCAGGCCT 57 5025 5044 161 590105 272 291ACATGGAATCCATGCAGGCC 52 5026 5045 162 590106 273 292AACATGGAATCCATGCAGGC 54 5027 5046 163 590107 274 293GAACATGGAATCCATGCAGG 51 5028 5047 164 590108 275 294TGAACATGGAATCCATGCAG 58 5029 5048 165 393340 276 295ATGAACATGGAATCCATGCA 62 5030 5049 166 590109 316 335GACCTGCACTGGTACAGCCT 69 7632 7651 167 436847 317 336GGACCTGCACTGGTACAGCC 74 7633 7652 168 590110 318 337AGGACCTGCACTGGTACAGC 70 7634 7653 169 590111 319 338GAGGACCTGCACTGGTACAG 74 7635 7654 170 590112 320 339TGAGGACCTGCACTGGTACA 68 7636 7655 171 590113 321 340GTGAGGACCTGCACTGGTAC 80 7637 7656 172 393343 322 341AGTGAGGACCTGCACTGGTA 79 7638 7657 173 590114 323 342AAGTGAGGACCTGCACTGGT 65 7639 7658 174 590115 324 343AAAGTGAGGACCTGCACTGG 48 7640 7659 175 590116 325 344TAAAGTGAGGACCTGCACTG 51 7641 7660 176 436848 326 345TTAAAGTGAGGACCTGCACT 59 7642 7661 177 590117 327 346ATTAAAGTGAGGACCTGCAC 43 7643 7662 178 590118 328 347GATTAAAGTGAGGACCTGCA 43 7644 7663 179 590119 329 348GGATTAAAGTGAGGACCTGC 67 7645 7664 180 590120 330 349AGGATTAAAGTGAGGACCTG 63 7646 7665 181 436849 331 350GAGGATTAAAGTGAGGACCT 64 7647 7666 182 393344 332 351AGAGGATTAAAGTGAGGACC 59 7648 7667 183 590121 333 352TAGAGGATTAAAGTGAGGAC 52 7649 7668 184 590122 334 353ATAGAGGATTAAAGTGAGGA 36 7650 7669 185 590123 335 354GATAGAGGATTAAAGTGAGG 25 7651 7670 186 590124 336 355GGATAGAGGATTAAAGTGAG 34 7652 7671 187 590125 337 356TGGATAGAGGATTAAAGTGA 49 7653 7672 188 590126 338 357CTGGATAGAGGATTAAAGTG 34 7654 7673 189 590127 339 358TCTGGATAGAGGATTAAAGT 39 7655 7674 190 590128 360 379ATCCTTTGGCCCACCGTGTT 60 7676 7695 191

TABLE 2 Percent inhibition of SOD-1 mRNA by 5-10-5 MOE gapmers targetingSEQ ID NO: 1 and/or 2 SEQ SEQ SEQ SEQ ID ID % % ID ID NO: 1 NO: 1inhibition inhibition NO: 2 NO: 2 SEQ Start Stop with with Start Stop IDISIS NO Site Site Sequence RTS3898 HTS90 Site Site NO 333611 167 186CCGTCGCCCTTCAGCACGCA 76 95 973 992 21 393347 361 380CATCCTTTGGCCCACCGTGT 70 72 7677 7696 192 590129 362 381TCATCCTTTGGCCCACCGTG 66 68 7678 7697 193 590130 363 382TTCATCCTTTGGCCCACCGT 53 55 7679 7698 194 590131 364 383CTTCATCCTTTGGCCCACCG 52 50 7680 7699 195 590132 365 384TCTTCATCCTTTGGCCCACC 61 64 7681 7700 196 590133 366 385CTCTTCATCCTTTGGCCCAC 45 54 7682 7701 197 590134 367 386TCTCTTCATCCTTTGGCCCA 44 34 7683 7702 198 590135 368 387CTCTCTTCATCCTTTGGCCC 52 49 7684 7703 199 590136 369 388CCTCTCTTCATCCTTTGGCC 48 47 7685 7704 200 590137 370 389GCCTCTCTTCATCCTTTGGC 35 44 n/a n/a 201 590138 371 390TGCCTCTCTTCATCCTTTGG 52 45 n/a n/a 202 590139 372 391ATGCCTCTCTTCATCCTTTG 50 45 n/a n/a 203 590140 373 392CATGCCTCTCTTCATCCTTT 49 27 n/a n/a 204 590141 374 393ACATGCCTCTCTTCATCCTT 34 18 n/a n/a 205 590142 375 394AACATGCCTCTCTTCATCCT 38 35 n/a n/a 206 333612 376 395CAACATGCCTCTCTTCATCC 34 33 n/a n/a 25 333613 377 396CCAACATGCCTCTCTTCATC 46 55 n/a n/a 26 333614 378 397TCCAACATGCCTCTCTTCAT 42 48 n/a n/a 27 333615 379 398CTCCAACATGCCTCTCTTCA 42 15 n/a n/a 28 333616 380 399TCTCCAACATGCCTCTCTTC 35 44 n/a n/a 29 333617 381 400GTCTCCAACATGCCTCTCTT 42 47 n/a n/a 30 590143 501 520TGCTTTTTCATGGACCACCA n.d. 65 n/a n/a 207 590144 502 521CTGCTTTTTCATGGACCACC n.d. 70 n/a n/a 208 590145 503 522TCTGCTTTTTCATGGACCAC n.d. 64 n/a n/a 209 436860 504 523ATCTGCTTTTTCATGGACCA n.d. 65 n/a n/a 210 590146 505 524CATCTGCTTTTTCATGGACC n.d. 68 9655 9674 211 590147 506 525TCATCTGCTTTTTCATGGAC n.d. 59 9656 9675 212 393359 507 526GTCATCTGCTTTTTCATGGA n.d. 56 9657 9676 213 590148 508 527AGTCATCTGCTTTTTCATGG n.d. 45 9658 9677 214 590149 509 528AAGTCATCTGCTTTTTCATG n.d. 23 9659 9678 215 590150 510 529CAAGTCATCTGCTTTTTCAT n.d. 43 9660 9679 216 590151 511 530CCAAGTCATCTGCTTTTTCA n.d. 72 9661 9680 217 489513 512 531CCCAAGTCATCTGCTTTTTC n.d. 73 9662 9681 218 590152 513 532GCCCAAGTCATCTGCTTTTT n.d. 74 9663 9682 219 436861 514 533TGCCCAAGTCATCTGCTTTT n.d. 75 9664 9683 220 590153 515 534TTGCCCAAGTCATCTGCTTT n.d. 47 9665 9684 221 393360 516 535TTTGCCCAAGTCATCTGCTT n.d. 57 9666 9685 222 590154 517 536CTTTGCCCAAGTCATCTGCT n.d. 79 9667 9686 223 590155 518 537CCTTTGCCCAAGTCATCTGC n.d. 67 9668 9687 224 590156 519 538ACCTTTGCCCAAGTCATCTG n.d. 57 9669 9688 225 333620 520 539CACCTTTGCCCAAGTCATCT n.d. 68 9670 9689 31 333621 521 540CCACCTTTGCCCAAGTCATC n.d. 72 9671 9690 32 333622 522 541TCCACCTTTGCCCAAGTCAT n.d. 77 9672 9691 33 333623 523 542TTCCACCTTTGCCCAAGTCA n.d. 73 9673 9692 34 333624 524 543TTTCCACCTTTGCCCAAGTC n.d. 77 9674 9693 35 333625 525 544ATTTCCACCTTTGCCCAAGT n.d. 79 9675 9694 36 333626 526 545CATTTCCACCTTTGCCCAAG n.d. 72 9676 9695 37 333627 527 546TCATTTCCACCTTTGCCCAA n.d. 55 9677 9696 38 333628 528 547TTCATTTCCACCTTTGCCCA n.d. 59 9678 9697 39 333629 529 548CTTCATTTCCACCTTTGCCC n.d. 73 9679 9698 40 333630 530 549TCTTCATTTCCACCTTTGCC n.d. 76 9680 9699 41 333631 531 550TTCTTCATTTCCACCTTTGC n.d. 62 9681 9700 42 333632 532 551TTTCTTCATTTCCACCTTTG n.d. 64 9682 9701 43 333633 533 552CTTTCTTCATTTCCACCTTT n.d. 69 9683 9702 44 333634 534 553ACTTTCTTCATTTCCACCTT n.d. 55 9684 9703 45 333635 535 554TACTTTCTTCATTTCCACCT n.d. 72 9685 9704 46 489517 582 601CCCAATTACACCACAAGCCA 68 72 9732 9751 226 436863 583 602TCCCAATTACACCACAAGCC 83 86 9733 9752 227 590157 584 603ATCCCAATTACACCACAAGC 64 62 9734 9753 228 590158 585 604GATCCCAATTACACCACAAG 51 61 9735 9754 229 590159 586 605CGATCCCAATTACACCACAA 60 55 9736 9755 230 590160 587 606GCGATCCCAATTACACCACA 59 63 9737 9756 231 150463 588 607GGCGATCCCAATTACACCAC 78 79 9738 9757 47 393363 589 608GGGCGATCCCAATTACACCA 65 65 9739 9758 232 590161 590 609TGGGCGATCCCAATTACACC 56 60 9740 9759 233 590162 591 610TTGGGCGATCCCAATTACAC 48 51 9741 9760 234 489518 592 611ATTGGGCGATCCCAATTACA 51 59 9742 9761 235 436864 593 612TATTGGGCGATCCCAATTAC 39 41 9743 9762 236 590163 594 613TTATTGGGCGATCCCAATTA 35 34 9744 9763 237 590164 595 614TTTATTGGGCGATCCCAATT 42 44 9745 9764 238 590165 596 615GTTTATTGGGCGATCCCAAT 58 61 9746 9765 239 393364 597 616TGTTTATTGGGCGATCCCAA 60 69 9747 9766 240 590166 598 617ATGTTTATTGGGCGATCCCA 51 54 9748 9767 241 590167 599 618AATGTTTATTGGGCGATCCC 48 45 9749 9768 242 590168 600 619GAATGTTTATTGGGCGATCC 60 65 9750 9769 243 150464 601 620GGAATGTTTATTGGGCGATC 58 63 9751 9770 48 393365 607 626TCCAAGGGAATGTTTATTGG 50 58 9757 9776 244

TABLE 3 Percent inhibition of SOD-1 mRNA by 5-10-5 MOE gapmers targetingSEQ ID NO: 1 and/or 2 SEQ SEQ SEQ SEQ ID ID % ID ID NO: 1 NO: 1inhibition NO: 2 NO: 2 SEQ ISIS Start Stop with Start Stop ID NO SiteSite Sequence RTS3898 Site Site NO 333611 167 186 CCGTCGCCCTTCAGCACGCA80 973 992 21 590169 608 627 ATCCAAGGGAATGTTTATTG 63 9758 9777 245590170 609 628 CATCCAAGGGAATGTTTATT 53 9759 9778 246 590171 610 629ACATCCAAGGGAATGTTTAT 49 9760 9779 247 590172 611 630TACATCCAAGGGAATGTTTA 56 9761 9780 248 489519 612 631CTACATCCAAGGGAATGTTT 60 9762 9781 249 590173 613 632ACTACATCCAAGGGAATGTT 61 9763 9782 250 590174 614 633GACTACATCCAAGGGAATGT 65 9764 9783 251 393366 615 634AGACTACATCCAAGGGAATG 58 9765 9784 252 590175 616 635CAGACTACATCCAAGGGAAT 50 9766 9785 253 436865 617 636TCAGACTACATCCAAGGGAA 69 9767 9786 254 590176 618 637CTCAGACTACATCCAAGGGA 78 9768 9787 255 150465 619 638CCTCAGACTACATCCAAGGG 91 9769 9788 49 590177 620 639 GCCTCAGACTACATCCAAGG90 9770 9789 256 590178 621 640 GGCCTCAGACTACATCCAAG 92 9771 9790 257489520 622 641 GGGCCTCAGACTACATCCAA 88 9772 9791 258 590179 643 662CAGGATAACAGATGAGTTAA 79 9793 9812 259 590180 644 663GCAGGATAACAGATGAGTTA 83 9794 9813 260 590181 645 664AGCAGGATAACAGATGAGTT 81 9795 9814 261 590182 646 665TAGCAGGATAACAGATGAGT 68 9796 9815 262 590183 647 666CTAGCAGGATAACAGATGAG 74 9797 9816 263 590184 648 667GCTAGCAGGATAACAGATGA 70 9798 9817 264 393370 649 668AGCTAGCAGGATAACAGATG 61 9799 9818 265 590185 650 669CAGCTAGCAGGATAACAGAT 78 9800 9819 266 590186 651 670ACAGCTAGCAGGATAACAGA 72 9801 9820 267 489522 652 671TACAGCTAGCAGGATAACAG 78 9802 9821 268 590187 653 672CTACAGCTAGCAGGATAACA 88 9803 9822 269 378879 654 673TCTACAGCTAGCAGGATAAC 86 9804 9823 270 590188 655 674TTCTACAGCTAGCAGGATAA 85 9805 9824 271 393371 656 675TTTCTACAGCTAGCAGGATA 84 9806 9825 272 436868 657 676ATTTCTACAGCTAGCAGGAT 81 9807 9826 273 590189 658 677CATTTCTACAGCTAGCAGGA 87 9808 9827 274 590190 659 678ACATTTCTACAGCTAGCAGG 92 9809 9828 275 590191 660 679TACATTTCTACAGCTAGCAG 88 9810 9829 276 590192 661 680ATACATTTCTACAGCTAGCA 88 9811 9830 277 489523 662 681GATACATTTCTACAGCTAGC 93 9812 9831 278 590193 683 702ACAGTGTTTAATGTTTATCA 74 9833 9852 279 590194 684 703TACAGTGTTTAATGTTTATC 64 9834 9853 280 590195 685 704TTACAGTGTTTAATGTTTAT 56 9835 9854 281 590196 686 705ATTACAGTGTTTAATGTTTA 50 9836 9855 282 590197 687 706GATTACAGTGTTTAATGTTT 74 9837 9856 283 590198 688 707AGATTACAGTGTTTAATGTT 37 9838 9857 284 590199 689 708AAGATTACAGTGTTTAATGT 58 9839 9858 285 393375 690 709TAAGATTACAGTGTTTAATG 58 9840 9859 286 590200 691 710TTAAGATTACAGTGTTTAAT 46 9841 9860 287 436876 772 791CAAATCTTCCAAGTGATCAT 36 9922 9941 288 590201 773 792ACAAATCTTCCAAGTGATCA 33 9923 9942 289 590202 774 793TACAAATCTTCCAAGTGATC 34 9924 9943 290 150474 775 794ATACAAATCTTCCAAGTGAT 47 9925 9944 50 590203 776 795 TATACAAATCTTCCAAGTGA29 9926 9945 291 393382 777 796 CTATACAAATCTTCCAAGTG 41 9927 9946 292436877 778 797 ACTATACAAATCTTCCAAGT 45 9928 9947 293 590204 779 798AACTATACAAATCTTCCAAG 27 9929 9948 294 590205 780 799AAACTATACAAATCTTCCAA 33 9930 9949 295 590206 781 800AAAACTATACAAATCTTCCA 35 9931 9950 296 489533 782 801TAAAACTATACAAATCTTCC 26 9932 9951 297 590207 783 802ATAAAACTATACAAATCTTC 19 9933 9952 298 590208 784 803TATAAAACTATACAAATCTT 2 9934 9953 299 590209 785 804 TTATAAAACTATACAAATCT7 9935 9954 300 590210 786 805 TTTATAAAACTATACAAATC 0 9936 9955 301590211 787 806 TTTTATAAAACTATACAAAT 4 9937 9956 302 590212 788 807GTTTTATAAAACTATACAAA 5 9938 9957 303 590213 789 808 AGTTTTATAAAACTATACAA3 9939 9958 304 436878 790 809 GAGTTTTATAAAACTATACA 7 9940 9959 305150475 791 810 TGAGTTTTATAAAACTATAC 28 9941 9960 51 489536 812 831CATTGAAACAGACATTTTAA 28 9962 9981 306 150479 813 832TCATTGAAACAGACATTTTA 36 9963 9982 52 393385 814 833 GTCATTGAAACAGACATTTT50 9964 9983 307 590214 815 834 GGTCATTGAAACAGACATTT 45 9965 9984 308590215 816 835 AGGTCATTGAAACAGACATT 47 9966 9985 309 590216 817 836CAGGTCATTGAAACAGACAT 39 9967 9986 310 590217 818 837ACAGGTCATTGAAACAGACA 44 9968 9987 311 590218 819 838TACAGGTCATTGAAACAGAC 42 9969 9988 312 150480 820 839ATACAGGTCATTGAAACAGA 46 9970 9989 53 393386 821 840 AATACAGGTCATTGAAACAG36 9971 9990 313 489537 822 841 AAATACAGGTCATTGAAACA 12 9972 9991 314590219 823 842 AAAATACAGGTCATTGAAAC 16 9973 9992 315 590220 824 843CAAAATACAGGTCATTGAAA 21 9974 9993 316

TABLE 4 Percent inhibition of SOD-1 mRNA by 5-10-5 MOE gapmers targetingSEQ ID NO: 1 and/or 2 SEQ SEQ SEQ SEQ ID ID % ID ID NO: 1 NO: 1inhibition NO: 2 NO: 2 SEQ ISIS Start Stop with Start Stop ID NO SiteSite Sequence RTS3898 Site Site NO 590251 n/a n/a CCTTGCCTTCTGCTCGAAAT57 1013 1032 317 590252 n/a n/a AATAAAGTTGACCTCTTTTT 45 5479 5498 318590253 n/a n/a CTCTGATATAAAAATCTTGT 54 8142 8161 319 590254 n/a n/aGCCCCGCGGCGGCCTCGGTC 38 1238 1257 320 590255 n/a n/aGCTATCGCCATTATTACAAG 38 7722 7741 321 590256 n/a n/aCTCAAATGTGAAAGTTGTCC 57 3414 3433 322 590257 n/a n/aGTTCTATATTCAATAAATGC 37 7925 7944 323 590258 n/a n/aAATTAAAGTTCCCAAATACA 0 7578 7597 324 590259 n/a n/a GATCATTACAAAAGTTAAGA17 6150 6169 325 590260 n/a n/a CCTTCTCTGCCCTTGCAGCC 55 1685 1704 326590261 n/a n/a ACCCAAATAACTATGTTGTA n.d. 9394 9413 327 590262 n/a n/aCCAGGTTTTAAACTTAACAA n.d. 8890 8909 328 590263 n/a n/aATCTCAGGACTAAAATAAAC 44 3663 3682 329 590264 n/a n/aAAATAACTATGTTGTAGACC n.d. 9390 9409 330 590265 n/a n/aAAGAACCTTTTCCAGAAAAT 37 2449 2468 331 590266 n/a n/aGGAACAGAAACAAGTCTATG 25 7458 7477 332 590267 n/a n/aAGAAAGCTATCGCCATTATT 27 7727 7746 333 590268 n/a n/aTTCCCAAATACATTCTAAAA 7 7570 7589 334 590269 n/a n/a AACTGCTCTAGGCCTGTGTC53 4787 4806 335 590270 n/a n/a AAATGGATCAAATCTGATCA 31 6595 6614 336590271 n/a n/a GTAGGTGCACATCAAAATCA 58 1928 1947 337 590272 n/a n/aTCTGATATAAAAATCTTGTC 28 8141 8160 338 590273 n/a n/aACCATATGAACTCCAGAAAG 45 7741 7760 339 590274 n/a n/aAACATCAAGGTAGTTCATGA 10 8379 8398 340 590275 n/a n/aGCAATTACAGAAATGGATCA 42 6605 6624 341 590276 n/a n/aTTTTAAGCATATTCCAAAGT 45 6331 6350 342 590277 n/a n/aTCAACCCCCAGCTCAAACAC 26 6174 6193 343 590278 n/a n/aAGAAAAATAACATTAATCCT n.d. 9541 9560 344 590279 n/a n/aAAGATTTTAAACACGGAATA 31 3085 3104 345 146145 165 184GTCGCCCTTCAGCACGCACA 82 971 990 54 333611 167 186 CCGTCGCCCTTCAGCACGCA81 973 992 21 590250 399 418 AGCAGTCACATTGCCCAAGT 75 8454 8473 346489525 682 701 CAGTGTTTAATGTTTATCAG 69 9832 9851 347 436879 825 844GCAAAATACAGGTCATTGAA 49 9975 9994 348 590221 826 845GGCAAAATACAGGTCATTGA 54 9976 9995 349 590222 827 846TGGCAAAATACAGGTCATTG 52 9977 9996 350 393387 828 847CTGGCAAAATACAGGTCATT 51 9978 9997 351 590223 829 848TCTGGCAAAATACAGGTCAT 47 9979 9998 352 590224 830 849GTCTGGCAAAATACAGGTCA 44 9980 9999 353 590225 831 850AGTCTGGCAAAATACAGGTC 50 9981 10000 354 489538 832 851AAGTCTGGCAAAATACAGGT 38 9982 10001 355 590226 833 852TAAGTCTGGCAAAATACAGG 33 9983 10002 356 590227 834 853TTAAGTCTGGCAAAATACAG 20 9984 10003 357 150482 853 872TTTAATACCCATCTGTGATT 29 10003 10022 55 590228 854 873GTTTAATACCCATCTGTGAT 33 10004 10023 358 150483 855 874AGTTTAATACCCATCTGTGA 44 10005 10024 56 590229 856 875AAGTTTAATACCCATCTGTG 51 10006 10025 359 590230 857 876CAAGTTTAATACCCATCTGT 42 10007 10026 360 590231 858 877ACAAGTTTAATACCCATCTG 38 10008 10027 361 393389 859 878GACAAGTTTAATACCCATCT 48 10009 10028 362 590232 860 879TGACAAGTTTAATACCCATC 55 10010 10029 363 590233 861 880CTGACAAGTTTAATACCCAT 49 10011 10030 364 489541 862 881TCTGACAAGTTTAATACCCA 52 10012 10031 365 590234 863 882TTCTGACAAGTTTAATACCC 39 10013 10032 366 590235 864 883ATTCTGACAAGTTTAATACC 21 10014 10033 367 590236 865 884AATTCTGACAAGTTTAATAC 4 10015 10034 368 393390 866 885AAATTCTGACAAGTTTAATA 7 10016 10035 369 590237 867 886GAAATTCTGACAAGTTTAAT 5 10017 10036 370 436881 868 887AGAAATTCTGACAAGTTTAA 33 10018 10037 371 590238 869 888AAGAAATTCTGACAAGTTTA 20 10019 10038 372 590239 891 910TTATTCACAGGCTTGAATGA 23 10041 10060 373 489544 892 911TTTATTCACAGGCTTGAATG 41 10042 10061 374 590240 893 912TTTTATTCACAGGCTTGAAT 40 10043 10062 375 436884 894 913TTTTTATTCACAGGCTTGAA 31 10044 10063 376 590241 895 914GTTTTTATTCACAGGCTTGA 39 10045 10064 377 150488 896 915GGTTTTTATTCACAGGCTTG 51 10046 10065 57 590242 897 916GGGTTTTTATTCACAGGCTT 46 10047 10066 378 150489 898 917AGGGTTTTTATTCACAGGCT 52 10048 10067 58 590243 899 918CAGGGTTTTTATTCACAGGC 49 10049 10068 379 590244 900 919ACAGGGTTTTTATTCACAGG 38 10050 10069 380 590245 901 920TACAGGGTTTTTATTCACAG 34 10051 10070 381 150490 902 921ATACAGGGTTTTTATTCACA 30 10052 10071 59 590246 903 922CATACAGGGTTTTTATTCAC 34 10053 10072 382 150491 904 923CCATACAGGGTTTTTATTCA 34 10054 10073 60 590247 905 924GCCATACAGGGTTTTTATTC 34 10055 10074 383 590248 906 925TGCCATACAGGGTTTTTATT 33 10056 10075 384 393393 907 926GTGCCATACAGGGTTTTTAT 43 10057 10076 385 590249 908 927AGTGCCATACAGGGTTTTTA 12 10058 10077 386

TABLE 5 Percent inhibition of SOD-1 mRNA by 5-10-5 MOE gapmers targetingSEQ ID NO: 1 and/or 2 SEQ SEQ SEQ SEQ ID ID % ID ID NO: 1 NO: 1inhibition NO: 2 NO: 2 SEQ ISIS Start Stop with Start Stop ID NO SiteSite Sequence RTS3898 Site Site NO 333611 167 186 CCGTCGCCCTTCAGCACGCA86 973 992 21 590280 n/a n/a TGGAAAAACTCAAATGTGAA 51 3422 3441 387590281 n/a n/a TTTCCCTTTCTTTTCCACAC 76 5738 5757 388 590282 n/a n/aTCTTTCCCTTTCTTTTCCAC 65 5740 5759 389 590283 n/a n/aTACCTTCTCTGCCCTTGCAG 74 1687 1706 390 590284 n/a n/aGCAAGGGCCAAGGCTGCTGC 75 6879 6898 391 590285 n/a n/aAAAGCTAAATTATGAATTAA 12 7592 7611 392 590286 n/a n/aCTAATGAAGGCTCAGTATGA 59 3193 3212 393 590287 n/a n/aGGAGTCAAATGCCAAAGAAC 60 2463 2482 394 590288 n/a n/aTGAATTAAAGTTCCCAAATA 5 7580 7599 395 590289 n/a n/a ACTTGGTGCAGGCAGAATAT63 6916 6935 396 590290 n/a n/a CCTCTGATATAAAAATCTTG 67 8143 8162 397590291 n/a n/a AAAGTTGGAGAGAGTTTCTG 8 4940 4959 398 590292 n/a n/aTCTCTGCCCTTGCAGCCCAA 80 1682 1701 399 590293 n/a n/aTTACTTGGTGCAGGCAGAAT 56 6918 6937 400 590294 n/a n/aAATGGAGTCAAATGCCAAAG 66 2466 2485 401 590295 n/a n/aTATGAATTAAAGTTCCCAAA 20 7582 7601 402 590296 n/a n/aAGTTCTATATTCAATAAATG 21 7926 7945 403 590297 n/a n/aTACAAGTAGTATACCATATG 33 7753 7772 404 590298 n/a n/aTAGCCTTAGAGCTGTACAAA 70 1553 1572 405 590299 n/a n/aGTCCCCATTTGTCAATTCCT 71 7882 7901 406 590300 n/a n/aAACCTGCCTACTGGCAGAGC 59 2095 2114 407 590301 n/a n/aCTTGTTCCCACACTCAATGC 56 4747 4766 408 590302 n/a n/aACAAGTCATGATAACCTGCA 61 8952 8971 409 590303 n/a n/aTGTTTTCCAAACTCAGATCT 52 8796 8815 410 590304 n/a n/aAGAACCTCATAATATTAGAA 9 9557 9576 411 590305 n/a n/a GGTTTTAAACTTAACAAAAT1 8887 8906 412 590306 n/a n/a CTCTGGTGTATTTTTAGTAA 65 1831 1850 413590307 n/a n/a TATCTCTGCATATCTGGAAA 71 3034 3053 414 590308 n/a n/aCAGCCTTTTTAACCCAAAAG 68 4407 4426 415 590309 n/a n/aTGGAATGCTCCACTATCCAA 57 3012 3031 416 590310 n/a n/aCGTTCAGAAGTTTGTCTCTG 67 2126 2145 417 590311 n/a n/aCTGCTCAGGGAAGGTGGAAA 53 2922 2941 418 590312 n/a n/aTCAAGAGAAGCTAGGAAAAC 50 3154 3173 419 590313 n/a n/aTCCCTTTCTTTTCCACACCT 74 5736 5755 420 590314 n/a n/aTTGTTCCCACACTCAATGCA 56 4746 4765 421 590315 n/a n/aTCACCAGCACAGCACAACAC 58 5076 5095 422 590316 n/a n/aCCTGGGATCATTACAAAAGT 42 6155 6174 423 590317 n/a n/aAGTAGTATACCATATGAACT 35 7749 7768 424 590318 n/a n/aTCTAATATGGTCAAATGTAA 27 8779 8798 425 590319 n/a n/aGGTTGGGCTCTGGTGTATTT 64 1838 1857 426 590320 n/a n/aTGCCCTTTACTTGGTGCAGG 56 6924 6943 427 590321 n/a n/aAGAGAGTTTCTGAACAAAGA 24 4932 4951 428 590322 n/a n/aGAATTTCAGCAATTACAGAA 33 6613 6632 429 590323 n/a n/aACAAGTTAAACAAGTCATGA 9 8961 8980 430 590324 n/a n/a TGTGCCCTTTACTTGGTGCA47 6926 6945 431 590325 n/a n/a TTAGGAGGAGGAAAAGGACC 23 1719 1738 432590326 n/a n/a ACTGGCAGAGCAATTTTAAA 25 2086 2105 433 590327 n/a n/aAGTCAAATGCCAAAGAACCT 58 2461 2480 434 590328 n/a n/aAAGCATCAGATGGATTAGGG 17 8411 8430 435 590329 n/a n/aGTCCGCGGGACCCTCAGGAA 54 1414 1433 436 590330 n/a n/aCAATTACAGAAATGGATCAA 42 6604 6623 437 590331 n/a n/aGCTGTCAAGTAATCACTACC 27 9606 9625 438 590332 n/a n/aAGTGCAAAGTTGGAGAGAGT 33 4945 4964 439 590333 n/a n/aACTTGCTTCCAATCCCAAAT 78 6436 6455 440 590334 n/a n/aAACTCAAATGTGAAAGTTGT 51 3416 3435 441 590335 n/a n/aTTTTAGTAAGATCTTCAAAT 14 1820 1839 442 590336 n/a n/aATTTCAGCAATTACAGAAAT 27 6611 6630 443 590337 n/a n/aTTAAGTGTCCCCATTTGTCA 56 7888 7907 444 590338 n/a n/aTTAGCAACCTGCCTACTGGC 57 2100 2119 445 590339 n/a n/aTATTACAAGAGTTAAGCATC 41 7711 7730 446 590340 n/a n/aATGTTGAATATACATGTACA 36 4545 4564 447 590341 n/a n/aTTTGTCTCTGACCATCTTAG 74 2116 2135 448 590342 n/a n/aTTTTCCACCAGTTGGTAACT 59 2253 2272 449 590343 n/a n/aCAACAGCTTCCCACAAGTTA 28 8973 8992 450 590344 n/a n/aCAAATGTGAAAGTTGTCCCT 62 3412 3431 451 590345 n/a n/aGCTACCTTCTCTGCCCTTGC 73 1689 1708 452 590346 n/a n/aTCTTAGCAGAACAGTGTTCT 51 8743 8762 453 590347 n/a n/aATACATTCTAAAAAGAAACA 41 7563 7582 454 590348 n/a n/aGCACATATTTACAAGTAGTA 58 7762 7781 455 590349 n/a n/aGGGTCACCAGCACAGCACAA 35 5079 5098 456 590350 n/a n/aGTGCAAGGGCCAAGGCTGCT 66 6881 6900 457 590351 n/a n/aACCTGGGTTCATGCATGGAT 72 2902 2921 458 590352 n/a n/aATCACTATTTGAAACTAAAT 0 6569 6588 459 590353 n/a n/a ATACAATAAAGTTGACCTCT64 5483 5502 460 590354 n/a n/a TTTTAAACTTAACAAAATGT 10 8885 8904 461590355 n/a n/a CTCCCCGCGCTCCCGCCACG 15 1268 1287 462 590356 n/a n/aGAAGGCTCAGTATGAAGAGA 65 3188 3207 463

TABLE 6 Percent inhibition of SOD-1 mRNA by 5-10-5 MOE gapmers targetingSEQ ID NO: 1 and/or 2 SEQ SEQ SEQ SEQ ID ID % ID ID NO: 1 NO: 1inhibition NO: 2 NO: 2 SEQ ISIS Start Stop with Start Stop ID NO SiteSite Sequence RTS3898 Site Site NO 333611 167 186 CCGTCGCCCTTCAGCACGCA87 973 992 21 590357 n/a n/a AGAAAACAGCTGATTTACCT 40 4915 4934 464590358 n/a n/a CCACAAGTTAAACAAGTCAT n.d. 8963 8982 465 590359 n/a n/aCAAATTTGCAAACAAGTAGC 61 8331 8350 466 590360 n/a n/aCCTAATTTGAACTGCAAGTA n.d. 8665 8684 467 590361 n/a n/aAAAAAACTCATCTCCCCAGC 70 6969 6988 468 590362 n/a n/aAGGCTCAGTATGAAGAGATC 67 3186 3205 469 590363 n/a n/aTGTTATCAAGAGCACAGGGC 58 3383 3402 470 590364 n/a n/aCCTCAAAAGGGAGATGGTAA 41 4768 4787 471 590365 n/a n/aAGTATGGGTCACCAGCACAG 71 5084 5103 472 590366 n/a n/aTCACAATCTAGTGCAGTTAC 70 5584 5603 473 590367 n/a n/aCAAGTGAGAAACCCAATCCT n.d. 8856 8875 474 590368 n/a n/aAGAAAATCTGGCCATTTTAA n.d. 8832 8851 475 590369 n/a n/aACAGGTAATGGTGCTCCGTG 71 3716 3735 476 590370 n/a n/aTGAAAGGCTTTCAGAAAACA 44 8102 8121 477 590371 n/a n/aCAGGCAAGTTACAGGAAGCA 64 6687 6706 478 590372 n/a n/aCAGCAAGCTGCTTAACTGCT 65 4800 4819 479 590373 n/a n/aTGTTGCAAAGACATTACCTT n.d. 9455 9474 480 590374 n/a n/aGAAACTAAATTAGCAAGATG 43 6559 6578 481 590375 n/a n/aTCAAGAGCACAGGGCCAAAA 60 3378 3397 482 590376 n/a n/aAGGAGGAGGAAAAGGACCTC 53 1717 1736 483 590377 n/a n/aCCTCAGCCTTTTTAACCCAA 73 4410 4429 484 590378 n/a n/aCTATGTTGTAGACCACCACA n.d. 9384 9403 485 590379 n/a n/aCTCCGTGGCTACATACAGAA 66 3703 3722 486 590380 n/a n/aTTTATCTGGATCTTTAGAAA n.d. 8642 8661 487 590381 n/a n/aAAAAAAAGGAAAGTGAAAGT n.d. 9279 9298 488 590382 n/a n/aGGTTCATGCATGGATTCTCA 76 2897 2916 489 590383 n/a n/aCTGCAAAGTGTCACACAAAC 76 1630 1649 490 590384 n/a n/aTTCAGAAGTACCAAAGGGTA 53 8227 8246 491 590385 n/a n/aTAAAAGCATTCCAGCATTTG 44 7848 7867 492 590386 n/a n/aTAGTATACCATATGAACTCC 73 7747 7766 493 590387 n/a n/aTGCATATCTGGAAAGCTGGA 59 3028 3047 494 590388 n/a n/aCTTAACTGCTCTAGGCCTGT 54 4790 4809 495 590389 n/a n/aAGGCACCGACCGGGCGGCAC 21 1155 1174 496 590390 n/a n/aTGCAAAGTTGGAGAGAGTTT 32 4943 4962 497 590391 n/a n/aTCCTCAAAAGGGAGATGGTA 37 4769 4788 498 590392 n/a n/aAGTATACCATATGAACTCCA 76 7746 7765 499 590393 n/a n/aTATTTGTACATGTTGAATAT  2 4554 4573 500 590394 n/a n/aACCCAAAAGGTGTATGTCTC 71 4396 4415 501 590395 n/a n/aCTTTGGAAAAAAAGGAAAGT n.d. 9285 9304 502 590396 n/a n/aGGGAGAAAGGCAGGCAAGTT 20 6697 6716 503 590397 n/a n/aTTAAGCCCAGGAAGTAAAAG  9 7862 7881 504 590398 n/a n/aAGACATTACCTTTAAACATT n.d. 9447 9466 505 590399 n/a n/aGTGGCTTAAGAAATGCTCCG 26 2050 2069 506 590400 n/a n/aGTGAGAAGGGAACAGAAACA 48 7466 7485 507 590401 n/a n/aAAAAGCATCAGATGGATTAG 21 8413 8432 508 590402 n/a n/aTTCCACCAGTTGGTAACTTC 78 2251 2270 509 590403 n/a n/aTTTTTAGTAAGATCTTCAAA 15 1821 1840 510 590404 n/a n/aATCTGTGTCCAAATCCCAGG 59 4847 4866 511 590405 n/a n/aTAAGATCTTCAAATAAGCTA 33 1814 1833 512 590406 n/a n/aATCAACTCTTTCCCTTTCTT 63 5746 5765 513 590407 n/a n/aTGTGTCCTCAAAAGGGAGAT 37 4773 4792 514 590408 n/a n/aTACCTCCTCCCAACAATACC n.d. 9590 9609 515 590409 n/a n/aTTCTGCTTTACAACTATGGC n.d. 9133 9152 516 590410 n/a n/aGTACATGTTGAATATACATG 35 4549 4568 517 590411 n/a n/aTTTGTGGCTAATCTTAAGGT 47 5699 5718 518 590412 n/a n/aTCCTGCCTCAGCCTTTTTAA 34 4415 4434 519 590413 n/a n/aCGGTGTCCGCGGGACCCTCA 59 1418 1437 520 590414 n/a n/aGAAATGGATCAAATCTGATC 50 6596 6615 521 590415 n/a n/aGGTAGTTCATGAGCTAAATT 31 8371 8390 522 590416 n/a n/aAATGGAGTCTCGACTAGTTT 62 8072 8091 523 590417 n/a n/aCAAGTATGGGTCACCAGCAC 57 5086 5105 524 590418 n/a n/aGGTGTCCGCGGGACCCTCAG 40 1417 1436 525 590419 n/a n/aCGCCACGCGCAGGCCCAGCC 37 1255 1274 526 590420 n/a n/aTCTAGGCCTGTGTCCTCAAA 75 4781 4800 527 590421 n/a n/aACTGTCCTGGGCTAATGAAG 36 3204 3223 528 590422 n/a n/aAAGCATCTTGTTACCTCTCT 52 7698 7717 529 590423 n/a n/aGCCCAGGAAGTAAAAGCATT 38 7858 7877 530 590424 n/a n/aGTAAGATCTTCAAATAAGCT 46 1815 1834 531 590425 n/a n/aAAAGGGAGATGGTAATCTTG 48 4763 4782 532 590426 n/a n/aGCCAAGGCTGCTGCCTTACA 66 6873 6892 533 590427 n/a n/aCAGACTAACTGTTCCTGTCC 43 2363 2382 534 590428 n/a n/aTTTGTCAATTCCTTTAAGCC 39 7875 7894 535 590429 n/a n/aACTACCTCCTCCCAACAATA n.d. 9592 9611 536 590430 n/a n/aTACCTCTCTTCATCCTTTGG 50 7687 7706 537 590431 n/a n/aACTGCTCTAGGCCTGTGTCC 59 4786 4805 538 590432 n/a n/aCCTCCTCCCAACAATACCCA n.d. 9588 9607 539 590433 n/a n/aGGCAGGCAAGTTACAGGAAG 42 6689 6708 540

TABLE 7 Percent inhibition of SOD-1 mRNA by 5-10-5 MOE gapmers targetingSEQ ID NO: 1 and/or 2 SEQ SEQ SEQ SEQ ID ID % ID ID NO: 1 NO: 1inhibition NO: 2 NO: 2 SEQ ISIS Start Stop with Start Stop ID NO SiteSite Sequence RTS3898 Site Site NO 592596 2 21 TCGCCCACTCTGGCCCCAAA 86808 827 541 592597 4 23 CCTCGCCCACTCTGGCCCCA 89 810 829 542 592598 6 25CGCCTCGCCCACTCTGGCCC 56 812 831 543 592599 8 27 CGCGCCTCGCCCACTCTGGC 68814 833 544 592600 10 29 TCCGCGCCTCGCCCACTCTG 64 816 835 545 592601 1231 CCTCCGCGCCTCGCCCACTC 83 818 837 546 592602 14 33 GACCTCCGCGCCTCGCCCAC89 820 839 547 592603 16 35 CAGACCTCCGCGCCTCGCCC 88 822 841 548 59260418 37 GCCAGACCTCCGCGCCTCGC 79 824 843 549 592605 20 39AGGCCAGACCTCCGCGCCTC 89 826 845 550 592606 22 41 ATAGGCCAGACCTCCGCGCC 88828 847 551 592607 24 43 TTATAGGCCAGACCTCCGCG 75 830 849 552 592608 2645 CTTTATAGGCCAGACCTCCG 21 832 851 553 592609 28 47 TACTTTATAGGCCAGACCTC76 834 853 554 592610 30 49 ACTACTTTATAGGCCAGACC 60 836 855 555 59261132 51 CGACTACTTTATAGGCCAGA 0 838 857 556 592612 34 53CGCGACTACTTTATAGGCCA 0 840 859 557 592613 36 55 TCCGCGACTACTTTATAGGC 0842 861 558 592614 38 57 TCTCCGCGACTACTTTATAG 7 844 863 559 592615 40 59CGTCTCCGCGACTACTTTAT 0 846 865 560 592616 42 61 CCCGTCTCCGCGACTACTTT 0848 867 561 592617 44 63 ACCCCGTCTCCGCGACTACT 0 850 869 562 592618 46 65GCACCCCGTCTCCGCGACTA 0 852 871 563 592619 48 67 CAGCACCCCGTCTCCGCGAC 0854 873 564 592620 50 69 ACCAGCACCCCGTCTCCGCG 0 856 875 565 592621 52 71AAACCAGCACCCCGTCTCCG 2 858 877 566 592622 54 73 GCAAACCAGCACCCCGTCTC 4860 879 567 592623 56 75 ACGCAAACCAGCACCCCGTC 0 862 881 568 592624 58 77CGACGCAAACCAGCACCCCG 0 864 883 569 592625 60 79 TACGACGCAAACCAGCACCC 0866 885 570 592626 62 81 ACTACGACGCAAACCAGCAC 0 868 887 571 592627 64 83AGACTACGACGCAAACCAGC 1 870 889 572 592628 66 85 GGAGACTACGACGCAAACCA 0872 891 573 592629 68 87 CAGGAGACTACGACGCAAAC 0 874 893 574 592630 70 89TGCAGGAGACTACGACGCAA 0 876 895 575 592631 72 91 GCTGCAGGAGACTACGACGC 1878 897 576 150511 74 93 ACGCTGCAGGAGACTACGAC 0 880 899 61 592632 90 109GCAACGGAAACCCCAGACGC 2 896 915 577 592633 92 111 CTGCAACGGAAACCCCAGAC 0898 917 578 592634 94 113 GACTGCAACGGAAACCCCAG 0 900 919 579 345715 95114 GGACTGCAACGGAAACCCCA 0 901 920 580 592635 96 115AGGACTGCAACGGAAACCCC 1 902 921 581 150437 98 117 CGAGGACTGCAACGGAAACC 6904 923 62 592636 100 119 TCCGAGGACTGCAACGGAAA 6 906 925 582 592637 102121 GTTCCGAGGACTGCAACGGA 12 908 927 583 592638 104 123TGGTTCCGAGGACTGCAACG 0 910 929 584 592639 106 125 CCTGGTTCCGAGGACTGCAA32 912 931 585 592640 108 127 GTCCTGGTTCCGAGGACTGC 68 914 933 586 345717110 129 AGGTCCTGGTTCCGAGGACT 65 916 935 587 592641 112 131CGAGGTCCTGGTTCCGAGGA 84 918 937 588 592642 114 133 GCCGAGGTCCTGGTTCCGAG86 920 939 589 592643 116 135 ACGCCGAGGTCCTGGTTCCG 78 922 941 590 592644118 137 CCACGCCGAGGTCCTGGTTC 79 924 943 591 345719 120 139GGCCACGCCGAGGTCCTGGT 63 926 945 592 150441 122 141 TAGGCCACGCCGAGGTCCTG81 928 947 63 592645 124 143 GCTAGGCCACGCCGAGGTCC 63 930 949 593 592646126 145 TCGCTAGGCCACGCCGAGGT 56 932 951 594 592647 128 147ACTCGCTAGGCCACGCCGAG 48 934 953 595 345721 130 149 TAACTCGCTAGGCCACGCCG63 936 955 596 592648 132 151 CATAACTCGCTAGGCCACGC 38 938 957 597 592649134 153 GCCATAACTCGCTAGGCCAC 52 940 959 598 592650 136 155TCGCCATAACTCGCTAGGCC 59 942 961 599 592651 138 157 CGTCGCCATAACTCGCTAGG55 944 963 600 592652 156 175 CAGCACGCACACGGCCTTCG 56 962 981 601 333605158 177 TTCAGCACGCACACGGCCTT 85 964 983 64 333606 160 179CCTTCAGCACGCACACGGCC 82 966 985 65 146144 162 181 GCCCTTCAGCACGCACACGG58 968 987 66 333609 164 183 TCGCCCTTCAGCACGCACAC 79 970 989 67 146145165 184 GTCGCCCTTCAGCACGCACA 86 971 990 54 333610 166 185CGTCGCCCTTCAGCACGCAC 79 972 991 68 333611 167 186 CCGTCGCCCTTCAGCACGCA83 973 992 21 592653 168 187 GCCGTCGCCCTTCAGCACGC 79 974 993 602 592654169 188 GGCCGTCGCCCTTCAGCACG 72 975 994 603 592655 170 189GGGCCGTCGCCCTTCAGCAC 51 976 995 604 592656 172 191 CTGGGCCGTCGCCCTTCAGC45 978 997 605 592657 174 193 CACTGGGCCGTCGCCCTTCA 33 980 999 606 592658176 195 TGCACTGGGCCGTCGCCCTT 72 982 1001 607 592659 178 197CCTGCACTGGGCCGTCGCCC 76 984 1003 608

TABLE 8 Percent inhibition of SOD-1 mRNA by 5-10-5 MOE gapmers targetingSEQ ID NO: 1 and/or 2 SEQ SEQ SEQ SEQ ID ID % ID ID NO: 1 NO: 1inhibition NO: 2 NO: 2 SEQ ISIS Start Stop with Start Stop ID NO SiteSite Sequence RTS3898 Site Site NO 333611 167 186 CCGTCGCCCTTCAGCACGCA87 973 992 21 150443 180 199 GCCCTGCACTGGGCCGTCGC 65 986 1005 69 592660182 201 ATGCCCTGCACTGGGCCGTC 52 988 1007 609 592661 184 203TGATGCCCTGCACTGGGCCG 30 990 1009 610 592662 186 205 GATGATGCCCTGCACTGGGC38 992 1011 611 592663 188 207 TTGATGATGCCCTGCACTGG 36 994 1013 612150444 190 209 AATTGATGATGCCCTGCACT 48 996 1015 70 592664 192 211GAAATTGATGATGCCCTGCA 35 998 1017 15 592665 194 213 TCGAAATTGATGATGCCCTG40 1000 1019 614 592666 196 215 GCTCGAAATTGATGATGCCC 68 1002 1021 615592667 198 217 CTGCTCGAAATTGATGATGC 63 1004 1023 616 592668 200 219TTCTGCTCGAAATTGATGAT 47 1006 1025 617 592669 239 258TTAATGCTTCCCCACACCTT 68 4993 5012 618 592670 241 260CTTTAATGCTTCCCCACACC 71 4995 5014 619 592671 243 262TCCTTTAATGCTTCCCCACA 69 4997 5016 620 150448 245 264AGTCCTTTAATGCTTCCCCA 76 4999 5018 71 592672 247 266 TCAGTCCTTTAATGCTTCCC75 5001 5020 621 592673 249 268 AGTCAGTCCTTTAATGCTTC 58 5003 5022 622592674 251 270 TCAGTCAGTCCTTTAATGCT 46 5005 5024 623 592675 253 272CTTCAGTCAGTCCTTTAATG 41 5007 5026 624 592676 255 274GCCTTCAGTCAGTCCTTTAA 62 5009 5028 625 150449 257 276AGGCCTTCAGTCAGTCCTTT 65 5011 5030 72 592677 259 278 GCAGGCCTTCAGTCAGTCCT69 5013 5032 626 592678 261 280 ATGCAGGCCTTCAGTCAGTC 65 5015 5034 627592679 263 282 CCATGCAGGCCTTCAGTCAG 53 5017 5036 628 592680 277 296CATGAACATGGAATCCATGC 63 5031 5050 629 592681 279 298CTCATGAACATGGAATCCAT 60 5033 5052 630 592682 281 300AACTCATGAACATGGAATCC 56 5035 5054 631 592683 284 303CCAAACTCATGAACATGGAA 60 5038 5057 632 592684 286 305CTCCAAACTCATGAACATGG 69 5040 5059 633 592685 288 307ATCTCCAAACTCATGAACAT 40 5042 5061 634 592686 290 309TTATCTCCAAACTCATGAAC 35 5044 5063 635 592687 292 311TATTATCTCCAAACTCATGA 26 5046 5065 636 592688 294 313TGTATTATCTCCAAACTCAT 41 5048 5067 637 150452 296 315GCTGTATTATCTCCAAACTC 51 5050 5069 73 592689 298 317 CTGCTGTATTATCTCCAAAC43 5052 5071 638 592690 300 319 GCCTGCTGTATTATCTCCAA 37 n/a n/a 639592691 302 321 CAGCCTGCTGTATTATCTCC 33 n/a n/a 640 592692 304 323TACAGCCTGCTGTATTATCT 27 n/a n/a 641 592693 306 325 GGTACAGCCTGCTGTATTAT21 n/a n/a 642 592694 308 327 CTGGTACAGCCTGCTGTATT 23 n/a n/a 643 592695310 329 CACTGGTACAGCCTGCTGTA 46 n/a n/a 644 592696 312 331TGCACTGGTACAGCCTGCTG 40 n/a n/a 645 592697 314 333 CCTGCACTGGTACAGCCTGC62 n/a n/a 646 592698 340 359 TTCTGGATAGAGGATTAAAG 41 7656 7675 647592699 342 361 TTTTCTGGATAGAGGATTAA 29 7658 7677 648 592700 344 363TGTTTTCTGGATAGAGGATT 51 7660 7679 649 592701 346 365CGTGTTTTCTGGATAGAGGA 64 7662 7681 650 592702 348 367ACCGTGTTTTCTGGATAGAG 44 7664 7683 651 592703 350 369CCACCGTGTTTTCTGGATAG 62 7666 7685 652 592704 352 371GCCCACCGTGTTTTCTGGAT 60 7668 7687 653 592705 354 373TGGCCCACCGTGTTTTCTGG 62 7670 7689 654 592706 356 375TTTGGCCCACCGTGTTTTCT 49 7672 7691 655 592707 358 377CCTTTGGCCCACCGTGTTTT 52 7674 7693 656 592708 382 401AGTCTCCAACATGCCTCTCT 53 n/a n/a 657 592709 384 403 CAAGTCTCCAACATGCCTCT39 n/a n/a 658 489501 386 405 CCCAAGTCTCCAACATGCCT 75 8441 8460 659150454 388 407 TGCCCAAGTCTCCAACATGC 86 8443 8462 74 592710 390 409ATTGCCCAAGTCTCCAACAT 71 8445 8464 660 592711 392 411ACATTGCCCAAGTCTCCAAC 64 8447 8466 661 592712 394 413TCACATTGCCCAAGTCTCCA 59 8449 8468 662 489502 396 415AGTCACATTGCCCAAGTCTC 70 8451 8470 663 592713 398 417GCAGTCACATTGCCCAAGTC 70 8453 8472 664 592714 400 419CAGCAGTCACATTGCCCAAG 84 8455 8474 665 592715 402 421GTCAGCAGTCACATTGCCCA 83 8457 8476 666 592716 404 423TTGTCAGCAGTCACATTGCC 59 8459 8478 667 489503 406 425CTTTGTCAGCAGTCACATTG 47 8461 8480 668 592717 408 427ATCTTTGTCAGCAGTCACAT 54 8463 8482 669 592718 410 429CCATCTTTGTCAGCAGTCAC 76 8465 8484 670 592719 412 431CACCATCTTTGTCAGCAGTC 75 8467 8486 671 592720 414 433CACACCATCTTTGTCAGCAG 66 8469 8488 672 489504 416 435GCCACACCATCTTTGTCAGC 60 8471 8490 673 592721 418 437CGGCCACACCATCTTTGTCA 62 8473 8492 674 592722 420 439ATCGGCCACACCATCTTTGT 57 8475 8494 675 592723 422 441ACATCGGCCACACCATCTTT 54 8477 8496 676 150458 424 443ACACATCGGCCACACCATCT 77 8479 8498 75 489505 426 445 AGACACATCGGCCACACCAT84 8481 8500 677 592724 428 447 ATAGACACATCGGCCACACC 66 8483 8502 678

TABLE 9 Percent inhibition of SOD-1 mRNA by 5-10-5 MOE gapmers targetingSEQ ID NO: 1 and/or 2 SEQ SEQ SEQ SEQ ID ID % % ID ID NO: 1 NO: 1inhibition inhibition NO: 2 NO: 2 SEQ ISIS Start Stop with with StartStop ID NO Site Site Sequence RTS3898 HTS90 Site Site NO 333611 167 186CCGTCGCCCTTCAGCACGCA 87 n.d. 973 992 21 592725 430 449CAATAGACACATCGGCCACA 67 65 8485 8504 679 592726 432 451TTCAATAGACACATCGGCCA 62 66 8487 8506 680 592727 434 453TCTTCAATAGACACATCGGC 56 49 8489 8508 681 489506 436 455AATCTTCAATAGACACATCG 25 28 8491 8510 682 592728 438 457AGAATCTTCAATAGACACAT 12 0 8493 8512 683 592729 440 459ACAGAATCTTCAATAGACAC 24 16 8495 8514 684 592730 442 461TCACAGAATCTTCAATAGAC 34 24 8497 8516 685 592731 444 463GATCACAGAATCTTCAATAG 15 14 8499 8518 686 489507 446 465GAGATCACAGAATCTTCAAT 42 46 8501 8520 687 592732 448 467GTGAGATCACAGAATCTTCA n.d. 58 8503 8522 688 592733 450 469GAGTGAGATCACAGAATCTT n.d. 45 8505 8524 689 592734 452 471GAGAGTGAGATCACAGAATC n.d. 48 8507 8526 690 592735 454 473CTGAGAGTGAGATCACAGAA n.d. 66 8509 8528 691 489508 456 475TCCTGAGAGTGAGATCACAG n.d. 60 8511 8530 692 333619 458 477TCTCCTGAGAGTGAGATCAC n.d. 65 8513 8532 76 592736 460 479GGTCTCCTGAGAGTGAGATC n.d. 40 8515 8534 693 592737 462 481ATGGTCTCCTGAGAGTGAGA n.d. 37 8517 8536 694 592738 464 483CAATGGTCTCCTGAGAGTGA n.d. 41 8519 8538 695 489509 466 485TGCAATGGTCTCCTGAGAGT n.d. 43 8521 8540 696 592739 468 487GATGCAATGGTCTCCTGAGA n.d. 16 8523 8542 697 592740 470 489ATGATGCAATGGTCTCCTGA n.d. 6 8525 8544 698 592741 472 491CAATGATGCAATGGTCTCCT n.d. 0 8527 8546 699 592742 474 493GCCAATGATGCAATGGTCTC n.d. 25 8529 8548 700 489510 476 495CGGCCAATGATGCAATGGTC n.d. 32 8531 8550 701 592743 478 497TGCGGCCAATGATGCAATGG n.d. 14 8533 8552 702 592744 480 499TGTGCGGCCAATGATGCAAT n.d. 0 8535 8554 703 592745 482 501AGTGTGCGGCCAATGATGCA n.d. 7 8537 8556 704 592746 484 503CCAGTGTGCGGCCAATGATG n.d. 25 8539 8558 705 489511 486 505CACCAGTGTGCGGCCAATGA n.d. 28 8541 8560 706 150460 488 507ACCACCAGTGTGCGGCCAAT n.d. 52 8543 8562 77 592747 490 509GGACCACCAGTGTGCGGCCA n.d. 44 n/a n/a 707 592748 492 511ATGGACCACCAGTGTGCGGC n.d. 40 n/a n/a 708 150462 494 513TCATGGACCACCAGTGTGCG n.d. 39 n/a n/a 78 592749 496 515TTTCATGGACCACCAGTGTG n.d. 35 n/a n/a 709 592750 498 517TTTTTCATGGACCACCAGTG n.d. 23 n/a n/a 710 592751 500 519GCTTTTTCATGGACCACCAG n.d. 63 n/a n/a 711 333636 536 555GTACTTTCTTCATTTCCACC n.d. 65 9686 9705 79 333638 538 557TTGTACTTTCTTCATTTCCA n.d. 66 9688 9707 80 333640 540 559CTTTGTACTTTCTTCATTTC n.d. 37 9690 9709 81 592752 543 562TGTCTTTGTACTTTCTTCAT n.d. 63 9693 9712 712 592753 545 564CCTGTCTTTGTACTTTCTTC n.d. 74 9695 9714 713 592754 547 566TTCCTGTCTTTGTACTTTCT n.d. 72 9697 9716 714 592755 549 568GTTTCCTGTCTTTGTACTTT n.d. 57 9699 9718 715 592756 568 587AAGCCAAACGACTTCCAGCG 72 66 9718 9737 716 592757 570 589ACAAGCCAAACGACTTCCAG 72 74 9720 9739 717 489516 572 591CCACAAGCCAAACGACTTCC 85 82 9722 9741 718 592758 574 593CACCACAAGCCAAACGACTT 72 73 9724 9743 719 592759 576 595TACACCACAAGCCAAACGAC 74 68 9726 9745 720 592760 578 597ATTACACCACAAGCCAAACG 67 61 9728 9747 721 592761 580 599CAATTACACCACAAGCCAAA 64 56 9730 9749 722 150466 640 659GATAACAGATGAGTTAAGGG 66 65 9790 9809 82 489521 642 661AGGATAACAGATGAGTTAAG 79 78 9792 9811 723 592762 663 682GGATACATTTCTACAGCTAG 91 87 9813 9832 724 592763 665 684CAGGATACATTTCTACAGCT 92 89 9815 9834 725 592764 667 686ATCAGGATACATTTCTACAG 88 83 9817 9836 726 592765 669 688TTATCAGGATACATTTCTAC 77 72 9819 9838 727 592766 671 690GTTTATCAGGATACATTTCT 90 89 9821 9840 728 592767 673 692ATGTTTATCAGGATACATTT 82 76 9823 9842 729 592768 675 694TAATGTTTATCAGGATACAT 80 79 9825 9844 730 592769 677 696TTTAATGTTTATCAGGATAC 82 78 9827 9846 731 592770 679 698TGTTTAATGTTTATCAGGAT 79 75 9829 9848 732 592771 681 700AGTGTTTAATGTTTATCAGG 84 81 9831 9850 733 489526 692 711TTTAAGATTACAGTGTTTAA 36 38 9842 9861 734 592772 694 713CTTTTAAGATTACAGTGTTT 46 47 9844 9863 735 592773 696 715CACTTTTAAGATTACAGTGT 39 42 9846 9865 736 592774 698 717TACACTTTTAAGATTACAGT 21 24 9848 9867 737 592775 700 719ATTACACTTTTAAGATTACA 3 0 9850 9869 738 489527 702 721CAATTACACTTTTAAGATTA 0 0 9852 9871 739 150467 704 723CACAATTACACTTTTAAGAT 58 73 9854 9873 83 592776 706 725CACACAATTACACTTTTAAG 29 5 9856 9875 740 592777 708 727GTCACACAATTACACTTTTA 59 49 9858 9877 741 592778 710 729AAGTCACACAATTACACTTT 40 34 9860 9879 742 489528 712 731AAAAGTCACACAATTACACT 31 27 9862 9881 743 592779 714 733GAAAAAGTCACACAATTACA 21 7 9864 9883 744 592780 716 735CTGAAAAAGTCACACAATTA 18 13 9866 9885 745 592781 718 737CTCTGAAAAAGTCACACAAT 32 26 9868 9887 746 592782 720 739AACTCTGAAAAAGTCACACA 35 20 9870 9889 747

TABLE 10 Percent inhibition of SOD-1 mRNA by 5-10-5 MOE gapmerstargeting SEQ ID NO: 1 and/or 2 SEQ SEQ SEQ SEQ ID ID % ID ID NO: 1 NO:1 inhibition NO: 2 NO: 2 SEQ ISIS Start Stop with Start Stop ID NO SiteSite Sequence RTS3898 Site Site NO 333611 167 186 CCGTCGCCCTTCAGCACGCA74 973 992 21 489529 722 741 GCAACTCTGAAAAAGTCACA 41 9872 9891 748592783 724 743 AAGCAACTCTGAAAAAGTCA 34 9874 9893 749 592784 727 746TTAAAGCAACTCTGAAAAAG 4 9877 9896 750 592785 729 748 CTTTAAAGCAACTCTGAAAA36 9879 9898 751 592786 731 750 TACTTTAAAGCAACTCTGAA 28 9881 9900 752592787 733 752 GGTACTTTAAAGCAACTCTG 48 9883 9902 753 592788 735 754CAGGTACTTTAAAGCAACTC 38 9885 9904 754 592789 737 756TACAGGTACTTTAAAGCAAC 20 9887 9906 755 592790 739 758ACTACAGGTACTTTAAAGCA 26 9889 9908 756 592791 741 760TCACTACAGGTACTTTAAAG 34 9891 9910 757 592792 743 762TCTCACTACAGGTACTTTAA 50 9893 9912 758 592793 745 764TTTCTCACTACAGGTACTTT 36 9895 9914 759 592794 747 766AGTTTCTCACTACAGGTACT 53 9897 9916 760 592795 749 768TCAGTTTCTCACTACAGGTA 37 9899 9918 761 150470 751 770AATCAGTTTCTCACTACAGG 30 9901 9920 84 592796 753 772 TAAATCAGTTTCTCACTACA21 9903 9922 762 150472 755 774 CATAAATCAGTTTCTCACTA 37 9905 9924 85592797 757 776 ATCATAAATCAGTTTCTCAC 35 9907 9926 763 592798 759 778TGATCATAAATCAGTTTCTC 35 9909 9928 764 592799 761 780AGTGATCATAAATCAGTTTC 5 9911 9930 765 592800 763 782 CAAGTGATCATAAATCAGTT21 9913 9932 766 592801 765 784 TCCAAGTGATCATAAATCAG 41 9915 9934 767592802 767 786 CTTCCAAGTGATCATAAATC 44 9917 9936 768 592803 769 788ATCTTCCAAGTGATCATAAA 30 9919 9938 769 592804 771 790AAATCTTCCAAGTGATCATA 32 9921 9940 770 489534 792 811CTGAGTTTTATAAAACTATA 4 9942 9961 771 150476 794 813 AACTGAGTTTTATAAAACTA9 9944 9963 86 592805 796 815 TTAACTGAGTTTTATAAAAC 14 9946 9965 772592806 798 817 TTTTAACTGAGTTTTATAAA 3 9948 9967 773 592807 800 819CATTTTAACTGAGTTTTATA 13 9950 9969 774 489535 802 821GACATTTTAACTGAGTTTTA 34 9952 9971 775 592808 804 823CAGACATTTTAACTGAGTTT 40 9954 9973 776 592809 806 825AACAGACATTTTAACTGAGT 36 9956 9975 777 592810 808 827GAAACAGACATTTTAACTGA 25 9958 9977 778 592811 810 829TTGAAACAGACATTTTAACT 24 9960 9979 779 592812 835 854TTTAAGTCTGGCAAAATACA 23 9985 10004 780 592813 837 856GATTTAAGTCTGGCAAAATA 31 9987 10006 781 592814 839 858GTGATTTAAGTCTGGCAAAA 41 9989 10008 782 592815 841 860CTGTGATTTAAGTCTGGCAA 49 9991 10010 783 592816 843 862ATCTGTGATTTAAGTCTGGC 53 9993 10012 784 150481 845 864CCATCTGTGATTTAAGTCTG 51 9995 10014 87 592817 847 866ACCCATCTGTGATTTAAGTC 51 9997 10016 785 592818 849 868ATACCCATCTGTGATTTAAG 43 9999 10018 786 592819 851 870TAATACCCATCTGTGATTTA 42 10001 10020 787 592820 870 889AAAGAAATTCTGACAAGTTT 22 10020 10039 788 489542 872 891ACAAAGAAATTCTGACAAGT 13 10022 10041 789 592821 874 893TGACAAAGAAATTCTGACAA 24 10024 10043 790 592822 876 895AATGACAAAGAAATTCTGAC 25 10026 10045 791 592823 878 897TGAATGACAAAGAAATTCTG 6 10028 10047 792 592824 880 899CTTGAATGACAAAGAAATTC 24 10030 10049 793 489543 882 901GGCTTGAATGACAAAGAAAT 29 10032 10051 794 592825 884 903CAGGCTTGAATGACAAAGAA 35 10034 10053 795 592826 886 905CACAGGCTTGAATGACAAAG 32 10036 10055 796 592827 888 907TTCACAGGCTTGAATGACAA 41 10038 10057 797 592828 890 909TATTCACAGGCTTGAATGAC 30 10040 10059 798 150492 909 928AAGTGCCATACAGGGTTTTT 32 10059 10078 88 592829 911 930ATAAGTGCCATACAGGGTTT 0 10061 10080 799 150493 913 932TAATAAGTGCCATACAGGGT 24 10063 10082 89 592830 915 934CATAATAAGTGCCATACAGG 26 10065 10084 800 150495 917 936CTCATAATAAGTGCCATACA 35 10067 10086 90 150496 919 938GCCTCATAATAAGTGCCATA 37 10069 10088 91 592831 921 940TAGCCTCATAATAAGTGCCA 19 10071 10090 801 592832 923 942AATAGCCTCATAATAAGTGC 0 10073 10092 802 592833 925 944TTAATAGCCTCATAATAAGT 19 10075 10094 803 150497 927 946TTTTAATAGCCTCATAATAA 17 10077 10096 92 592834 929 948TCTTTTAATAGCCTCATAAT 27 10079 10098 804 592835 931 950ATTCTTTTAATAGCCTCATA 27 10081 10100 805 150498 933 952GGATTCTTTTAATAGCCTCA 39 10083 10102 93 592836 935 954TTGGATTCTTTTAATAGCCT 24 10085 10104 806 592837 937 956ATTTGGATTCTTTTAATAGC 0 10087 10106 807 592838 939 958GAATTTGGATTCTTTTAATA 10 10089 10108 808 592839 941 960TTGAATTTGGATTCTTTTAA 13 10091 10110 809 592840 943 962GTTTGAATTTGGATTCTTTT 29 10093 10112 810 592841 945 964TAGTTTGAATTTGGATTCTT 31 10095 10114 811 592842 947 966TTTAGTTTGAATTTGGATTC 8 10097 10116 812 592843 949 968TTTTTAGTTTGAATTTGGAT 10 n/a n/a 813 592844 951 970 TTTTTTTAGTTTGAATTTGG7 n/a n/a 814

Example 2: Inhibition of Human SOD-1 in HepG2 Cells by MOE Gapmers

Modified oligonucleotides were designed targeting a superoxide dismutase1, soluble (SOD-1) nucleic acid and were tested for their effects onSOD-1 mRNA in vitro. ISIS 146143, ISIS 150438-150440, ISIS 150442, ISIS150450, ISIS 150455-150457, ISIS 150459, ISIS 150461, ISIS 150469, ISIS150473, ISIS 150478, ISIS 150484, ISIS 150486, ISIS 150494, ISIS150508-150510, ISIS 333607, ISIS 333608, ISIS 333611, ISIS 333618,previously disclosed in WO 2005/040180, were also included in thisassay. The modified oligonucleotides were tested in a series ofexperiments that had similar culture conditions. The results for eachexperiment are presented in separate tables shown below. Cultured HepG2cells at a density of 20,000 cells per well were transfected usingelectroporation with 5,000 nM modified oligonucleotide. After atreatment period of approximately 24 hours, RNA was isolated from thecells and SOD-1 mRNA levels were measured by quantitative real-time PCR.

Human primer probe set RTS3898 was used to measure mRNA levels. In caseswhere the oligonucleotide overlapped the amplicon of the primer probeset, an alternative primer probe set, HTS90, was used to measure mRNAlevels. SOD-1 mRNA levels were adjusted according to total RNA content,as measured by RIBOGREEN®. Results are presented as percent inhibitionof SOD-1, relative to untreated control cells. ‘n.d.’ indicates thatinhibition levels were not measured using the particular primer probeset.

The newly designed modified oligonucleotides in the Tables below weredesigned as 5-10-5 MOE gapmers. The 5-10-5 MOE gapmers are 20nucleosides in length, wherein the central gap segment is comprised often 2′-deoxyribonucleosides and is flanked by wing segments on the 5′direction and the 3′ directions comprising five nucleosides each. Eachnucleoside in the 5′ wing segment and each nucleoside in the 3′ wingsegment has a 2′-MOE modification. The internucleoside linkagesthroughout each gapmer are phosphorothioate linkages. All cytosineresidues throughout each gapmer are 5-methylcytosines. “Start site”indicates the 5′-most nucleoside to which the gapmer is targeted in thehuman gene sequence. “Stop site” indicates the 3′-most nucleoside towhich the gapmer is targeted human gene sequence. Each gapmer listed inthe Tables below is targeted to either the human SOD-1 mRNA, designatedherein as SEQ ID NO: 1 (GENBANK Accession No. NM_000454.4) or the humanSOD-1 genomic sequence, designated herein as SEQ ID NO: 2 (GENBANKAccession No. NT_011512.10 truncated from nucleotides 18693000 to Ser.No. 18/704,000). ‘n/a’ indicates that the modified oligonucleotide doesnot target that particular gene sequence with 100% complementarity.

TABLE 11 Percent inhibition of SOD-1 mRNA by 5-10-5 MOE gapmerstargeting SEQ ID NO: 1 and/or 2 SEQ SEQ SEQ SEQ ID ID % ID ID NO: 1 NO:1 inhibition NO: 2 NO: 2 SEQ ISIS Start Stop with Start Stop ID NO SiteSite Sequence RTS3898 Site Site NO 333611 167 186 CCGTCGCCCTTCAGCACGCA64 973 992 21 596301 550 569 CGTTTCCTGTCTTTGTACTT n.d. 9700 9719 815596302 569 588 CAAGCCAAACGACTTCCAGC 54 9719 9738 816 596303 571 590CACAAGCCAAACGACTTCCA 47 9721 9740 817 596304 573 592ACCACAAGCCAAACGACTTC 28 9723 9742 818 596305 575 594ACACCACAAGCCAAACGACT 49 9725 9744 819 596306 577 596TTACACCACAAGCCAAACGA 24 9727 9746 820 596307 641 660GGATAACAGATGAGTTAAGG 48 9791 9810 821 596308 664 683AGGATACATTTCTACAGCTA 79 9814 9833 822 596309 666 685TCAGGATACATTTCTACAGC 70 9816 9835 823 596310 668 687TATCAGGATACATTTCTACA 58 9818 9837 824 489524 672 691TGTTTATCAGGATACATTTC 52 9822 9841 825 596311 674 693AATGTTTATCAGGATACATT 54 9824 9843 826 596312 676 695TTAATGTTTATCAGGATACA 34 9826 9845 827 596313 678 697GTTTAATGTTTATCAGGATA 71 9828 9847 828 596314 680 699GTGTTTAATGTTTATCAGGA 73 9830 9849 829 596315 693 712TTTTAAGATTACAGTGTTTA 13 9843 9862 830 596316 695 714ACTTTTAAGATTACAGTGTT 24 9845 9864 831 596317 697 716ACACTTTTAAGATTACAGTG 15 9847 9866 832 596318 699 718TTACACTTTTAAGATTACAG 0 9849 9868 833 596319 701 720 AATTACACTTTTAAGATTAC1 9851 9870 834 596320 705 724 ACACAATTACACTTTTAAGA 0 9855 9874 835596321 707 726 TCACACAATTACACTTTTAA 15 9857 9876 836 596322 711 730AAAGTCACACAATTACACTT 15 9861 9880 837 596323 715 734TGAAAAAGTCACACAATTAC 0 9865 9884 838 596324 717 736 TCTGAAAAAGTCACACAATT5 9867 9886 839 596325 719 738 ACTCTGAAAAAGTCACACAA 21 9869 9888 840596326 723 742 AGCAACTCTGAAAAAGTCAC 14 9873 9892 841 596327 730 749ACTTTAAAGCAACTCTGAAA 0 9880 9899 842 489530 732 751 GTACTTTAAAGCAACTCTGA22 9882 9901 843 596328 734 753 AGGTACTTTAAAGCAACTCT 36 9884 9903 844596329 740 759 CACTACAGGTACTTTAAAGC 18 9890 9909 845 150469 742 761CTCACTACAGGTACTTTAAA 25 9892 9911 94 596330 744 763 TTCTCACTACAGGTACTTTA28 9894 9913 846 596331 746 765 GTTTCTCACTACAGGTACTT 30 9896 9915 847596332 748 767 CAGTTTCTCACTACAGGTAC 25 9898 9917 848 596333 750 769ATCAGTTTCTCACTACAGGT 22 9900 9919 849 489531 752 771AAATCAGTTTCTCACTACAG 0 9902 9921 850 596334 756 775 TCATAAATCAGTTTCTCACT21 9906 9925 851 596335 760 779 GTGATCATAAATCAGTTTCT 37 9910 9929 852489532 762 781 AAGTGATCATAAATCAGTTT 8 9912 9931 853 596336 764 783CCAAGTGATCATAAATCAGT 39 9914 9933 854 436935 766 785TTCCAAGTGATCATAAATCA 18 9916 9935 855 596337 768 787TCTTCCAAGTGATCATAAAT 12 9918 9937 856 150473 770 789AATCTTCCAAGTGATCATAA 4 9920 9939 95 596338 795 814 TAACTGAGTTTTATAAAACT0 9945 9964 857 596339 807 826 AAACAGACATTTTAACTGAG 4 9957 9976 858596340 809 828 TGAAACAGACATTTTAACTG 0 9959 9978 859 150478 811 830ATTGAAACAGACATTTTAAC 0 9961 9980 96 596341 836 855 ATTTAAGTCTGGCAAAATAC16 9986 10005 860 596342 840 859 TGTGATTTAAGTCTGGCAAA 34 9990 10009 861489539 842 861 TCTGTGATTTAAGTCTGGCA 44 9992 10011 862 596343 844 863CATCTGTGATTTAAGTCTGG 29 9994 10013 863 596344 846 865CCCATCTGTGATTTAAGTCT 41 9996 10015 864 596345 848 867TACCCATCTGTGATTTAAGT 50 9998 10017 865 596346 850 869AATACCCATCTGTGATTTAA 0 10000 10019 866 489540 852 871TTAATACCCATCTGTGATTT 11 10002 10021 867 150484 871 890CAAAGAAATTCTGACAAGTT 7 10021 10040 97 596347 873 892GACAAAGAAATTCTGACAAG 8 10023 10042 868 596348 877 896GAATGACAAAGAAATTCTGA 0 10027 10046 869 596349 883 902AGGCTTGAATGACAAAGAAA 27 10033 10052 870 150486 885 904ACAGGCTTGAATGACAAAGA 19 10035 10054 98 596350 910 929TAAGTGCCATACAGGGTTTT 13 10060 10079 871 596351 914 933ATAATAAGTGCCATACAGGG 18 10064 10083 872 150494 916 935TCATAATAAGTGCCATACAG 0 10066 10085 99 596352 918 937CCTCATAATAAGTGCCATAC 23 10068 10087 873 596353 920 939AGCCTCATAATAAGTGCCAT 6 10070 10089 874 596354 922 941ATAGCCTCATAATAAGTGCC 19 10072 10091 875 596355 928 947CTTTTAATAGCCTCATAATA 0 10078 10097 876 596356 930 949TTCTTTTAATAGCCTCATAA 5 10080 10099 877 596357 932 951GATTCTTTTAATAGCCTCAT 4 10082 10101 878 596358 934 953TGGATTCTTTTAATAGCCTC 13 10084 10103 879 596359 936 955TTTGGATTCTTTTAATAGCC 14 10086 10105 880 596360 938 957AATTTGGATTCTTTTAATAG 14 10088 10107 881 596361 940 959TGAATTTGGATTCTTTTAAT 0 10090 10109 882 596362 946 965TTAGTTTGAATTTGGATTCT 0 10096 10115 883 596363 948 967TTTTAGTTTGAATTTGGATT 0 n/a n/a 884 596364 950 969 TTTTTTAGTTTGAATTTGGA 0n/a n/a 885

TABLE 12 Percent inhibition of SOD-1 mRNA by 5-10-5 MOE gapmerstargeting SEQ ID NO: 1 and/or 2 SEQ SEQ SEQ SEQ ID ID % % ID ID NO: 1NO: 1 inhibition inhibition NO: 2 NO: 2 SEQ ISIS Start Stop with withStart Stop ID NO Site Site Sequence RTS3898 HTS90 Site Site NO 333611167 186 CCGTCGCCCTTCAGCACGCA 66 n.d. 973 992 21 596230 246 265CAGTCCTTTAATGCTTCCCC 51 40 5000 5019 886 596231 248 267GTCAGTCCTTTAATGCTTCC 34 34 5002 5021 887 596232 250 269CAGTCAGTCCTTTAATGCTT 35 29 5004 5023 888 596233 252 271TTCAGTCAGTCCTTTAATGC 24 21 5006 5025 889 596234 256 275GGCCTTCAGTCAGTCCTTTA 41 39 5010 5029 890 150450 258 277CAGGCCTTCAGTCAGTCCTT 56 51 5012 5031 100 596235 260 279TGCAGGCCTTCAGTCAGTCC 42 46 5014 5033 891 596236 262 281CATGCAGGCCTTCAGTCAGT 37 33 5016 5035 892 596237 278 297TCATGAACATGGAATCCATG 24 19 5032 5051 893 596238 280 299ACTCATGAACATGGAATCCA 27 20 5034 5053 894 596239 295 314CTGTATTATCTCCAAACTCA 32 28 5049 5068 895 596240 309 328ACTGGTACAGCCTGCTGTAT 22 28 n/a n/a 896 596241 311 330GCACTGGTACAGCCTGCTGT 31 24 n/a n/a 897 596242 313 332CTGCACTGGTACAGCCTGCT 38 29 n/a n/a 898 596243 315 334ACCTGCACTGGTACAGCCTG 46 48 n/a n/a 899 596244 341 360TTTCTGGATAGAGGATTAAA 6 14 7657 7676 900 596245 343 362GTTTTCTGGATAGAGGATTA 28 39 7659 7678 901 596246 347 366CCGTGTTTTCTGGATAGAGG 44 37 7663 7682 902 596247 349 368CACCGTGTTTTCTGGATAGA 24 11 7665 7684 903 596248 351 370CCCACCGTGTTTTCTGGATA 46 40 7667 7686 904 596249 353 372GGCCCACCGTGTTTTCTGGA 46 41 7669 7688 905 596250 355 374TTGGCCCACCGTGTTTTCTG 35 26 7671 7690 906 596251 357 376CTTTGGCCCACCGTGTTTTC 31 15 7673 7692 907 596252 359 378TCCTTTGGCCCACCGTGTTT 30 23 7675 7694 908 596253 383 402AAGTCTCCAACATGCCTCTC 20 6 n/a n/a 909 596254 387 406GCCCAAGTCTCCAACATGCC 61 53 8442 8461 910 596255 389 408TTGCCCAAGTCTCCAACATG 41 33 8444 8463 911 596256 391 410CATTGCCCAAGTCTCCAACA 39 25 8446 8465 912 150455 393 412CACATTGCCCAAGTCTCCAA 36 19 8448 8467 101 596257 397 416CAGTCACATTGCCCAAGTCT 40 27 8452 8471 913 596258 401 420TCAGCAGTCACATTGCCCAA 52 42 8456 8475 914 596259 403 422TGTCAGCAGTCACATTGCCC 55 49 8458 8477 915 596260 405 424TTTGTCAGCAGTCACATTGC 26 16 8460 8479 916 596261 407 426TCTTTGTCAGCAGTCACATT 20 11 8462 8481 917 596262 409 428CATCTTTGTCAGCAGTCACA 34 13 8464 8483 918 596263 411 430ACCATCTTTGTCAGCAGTCA 41 30 8466 8485 919 596264 415 434CCACACCATCTTTGTCAGCA 39 20 8470 8489 920 596265 417 436GGCCACACCATCTTTGTCAG 23 5 8472 8491 921 150456 419 438TCGGCCACACCATCTTTGTC 32 28 8474 8493 102 150457 421 440CATCGGCCACACCATCTTTG 34 38 8476 8495 103 596266 423 442CACATCGGCCACACCATCTT 27 13 8478 8497 922 596267 425 444GACACATCGGCCACACCATC 45 30 8480 8499 923 150459 427 446TAGACACATCGGCCACACCA 46 36 8482 8501 104 596268 429 448AATAGACACATCGGCCACAC 30 25 8484 8503 924 596269 431 450TCAATAGACACATCGGCCAC 35 0 8486 8505 925 596270 433 452CTTCAATAGACACATCGGCC 39 16 8488 8507 926 596271 435 454ATCTTCAATAGACACATCGG 16 0 8490 8509 927 596272 437 456GAATCTTCAATAGACACATC 22 11 8492 8511 928 596273 439 458CAGAATCTTCAATAGACACA 17 0 8494 8513 929 596274 441 460CACAGAATCTTCAATAGACA 10 14 8496 8515 930 596275 443 462ATCACAGAATCTTCAATAGA 11 10 8498 8517 931 596276 445 464AGATCACAGAATCTTCAATA 14 29 8500 8519 932 596277 447 466TGAGATCACAGAATCTTCAA n.d. 30 8502 8521 933 596278 449 468AGTGAGATCACAGAATCTTC n.d. 30 8504 8523 934 596279 453 472TGAGAGTGAGATCACAGAAT n.d. 18 8508 8527 935 333618 457 476CTCCTGAGAGTGAGATCACA n.d. 27 8512 8531 105 596281 459 478GTCTCCTGAGAGTGAGATCA n.d. 23 8514 8533 936 596282 461 480TGGTCTCCTGAGAGTGAGAT n.d. 24 8516 8535 937 596283 463 482AATGGTCTCCTGAGAGTGAG n.d. 22 8518 8537 938 596284 465 484GCAATGGTCTCCTGAGAGTG n.d. 57 8520 8539 939 596285 467 486ATGCAATGGTCTCCTGAGAG n.d. 0 8522 8541 940 596286 469 488TGATGCAATGGTCTCCTGAG n.d. 1 8524 8543 941 596287 471 490AATGATGCAATGGTCTCCTG n.d. 0 8526 8545 942 596288 473 492CCAATGATGCAATGGTCTCC n.d. 8 8528 8547 943 596289 475 494GGCCAATGATGCAATGGTCT n.d. 9 8530 8549 944 596290 477 496GCGGCCAATGATGCAATGGT n.d. 13 8532 8551 945 596291 479 498GTGCGGCCAATGATGCAATG n.d. 12 8534 8553 946 596292 481 500GTGTGCGGCCAATGATGCAA n.d. 15 8536 8555 947 596293 483 502CAGTGTGCGGCCAATGATGC n.d. 0 8538 8557 948 596294 485 504ACCAGTGTGCGGCCAATGAT n.d. 0 8540 8559 949 596295 487 506CCACCAGTGTGCGGCCAATG n.d. 22 8542 8561 950 596296 489 508GACCACCAGTGTGCGGCCAA n.d. 16 n/a n/a 951 596297 491 510TGGACCACCAGTGTGCGGCC n.d. 28 n/a n/a 952 150461 493 512CATGGACCACCAGTGTGCGG n.d. 25 n/a n/a 106 596298 495 514TTCATGGACCACCAGTGTGC n.d. 21 n/a n/a 953 596299 497 516TTTTCATGGACCACCAGTGT n.d. 17 n/a n/a 954 596300 499 518CTTTTTCATGGACCACCAGT n.d. 9 n/a n/a 955

Example 3: Inhibition of Human SOD-1 in HepG2 Cells by Deoxy, MOE andcEt Gapmers

Modified oligonucleotides were designed targeting a superoxide dismutase1, soluble (SOD-1) nucleic acid and were tested for their effects onSOD-1 mRNA in vitro. ISIS 333611, which was previously described in WO2005/040180, was included as a benchmark. ISIS 590067, ISIS 590074, ISIS590082, ISIS 590130, ISIS 590138, and ISIS 590146, which are 5-10-5 MOEgapmers as described above in Example 1, were also included in thisassay. ISIS 590512, which has a similar sequence as ISIS 333611 but withdeoxy, MOE, and cEt sugar modifications, was also included in thisstudy.

The modified oligonucleotides were tested in a series of experimentsthat had similar culture conditions. The results for each experiment arepresented in separate tables shown below. Cultured HepG2 cells at adensity of 20,000 cells per well were transfected using electroporationwith 3,000 nM modified oligonucleotide. After a treatment period ofapproximately 24 hours, RNA was isolated from the cells and SOD-1 mRNAlevels were measured by quantitative real-time PCR.

Human primer probe set RTS3898 was used to measure mRNA levels. SOD-1mRNA levels were adjusted according to total RNA content, as measured byRIBOGREEN®. Results are presented as percent inhibition of SOD-1,relative to untreated control cells. ‘n.d.’ indicates that inhibitionlevels were not measured.

The newly designed modified oligonucleotides in the Tables below weredesigned as deoxy, MOE, and cEt gapmers. The gapmers are 17 nucleosidesin length wherein each nucleoside has a MOE sugar modification, a cEtsugar modification, or a deoxy moiety. The sugar chemistry of eacholigonucleotide is denoted as in the Chemistry column, where ‘k’indicates a cEt modified sugar; ‘d’ indicates a 2′-deoxyribose; and ‘e’indicates a 2′-MOE modified sugar. The internucleoside linkagesthroughout each gapmer are phosphorothioate linkages. All cytosineresidues throughout each gapmer are 5-methylcytosines. “Start site”indicates the 5′-most nucleoside to which the gapmer is targeted in thehuman gene sequence. “Stop site” indicates the 3′-most nucleoside towhich the gapmer is targeted human gene sequence. Each gapmer listed inthe Tables below is targeted to either the human SOD-1 mRNA, designatedherein as SEQ ID NO: 1 (GENBANK Accession No. NM_000454.4) or the humanSOD-1 genomic sequence, designated herein as SEQ ID NO: 2 (GENBANKAccession No. NT_011512.10 truncated from nucleotides 18693000 to Ser.No. 18/704,000). ‘n/a’ indicates that the modified oligonucleotide doesnot target that particular gene sequence with 100% complementarity.

TABLE 13 Percent inhibition of SOD-1 mRNA by deoxy, MOE and cEt gapmerstargeting SEQ ID NO: 1 and/or 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1NO: 2 NO: 2 SEQ ISIS Start Stop % Start Stop ID NO Site Site SequenceChemistry inhibition Site Site NO 590434 1 17 CCACTCTGGCCCeeekkdddddddkkeee 17 807 823 956 CAAAC 590435 2 18 CCCACTCTGGCCeeekkdddddddkkeee 15 808 824 957 CCAAA 590436 3 19 GCCCACTCTGGCeeekkdddddddkkeee 22 809 825 958 CCCAA 590437 4 20 CGCCCACTCTGGeeekkdddddddkkeee 14 810 826 959 CCCCA 590438 35 51 CGACTACTTTATeeekkdddddddkkeee 12 841 857 960 AGGCC 590439 36 52 GCGACTACTTTAeeekkdddddddkkeee 12 842 858 961 TAGGC 590440 37 53 CGCGACTACTTTeeekkdddddddkkeee 11 843 859 962 ATAGG 590441 38 54 CCGCGACTACTTeeekkdddddddkkeee 5 844 860 963 TATAG 590442 76 92 CGCTGCAGGAGAeeekkdddddddkkeee 0 882 898 964 CTACG 590443 77 93 ACGCTGCAGGAGeeekkdddddddkkeee 25 883 899 965 ACTAC 590444 167 183 TCGCCCTTCAGCeeekkdddddddkkeee 31 973 989 966 ACGCA 590445 168 184 GTCGCCCTTCAGeeekkdddddddkkeee 28 974 990 967 CACGC 590512 169 185 CGTCGCCCTTCAeeekkdddddddkkeee 8 975 991 968 GCACG 590446 170 186 CCGTCGCCCTTCeeekkdddddddkkeee 27 976 992 969 AGCAC 590447 171 187 GCCGTCGCCCTTeeekkdddddddkkeee 33 977 993 970 CAGCA 590448 202 218 TCTGCTCGAAATeeekkdddddddkkeee 34 1008 1024 971 TGATG 590449 203 219 TTCTGCTCGAAAeeekkdddddddkkeee 18 1009 1025 972 TTGAT 590450 204 220 CTTCTGCTCGAAeeekkdddddddkkeee 13 1010 1026 973 ATTGA 590451 205 221 CCTTCTGCTCGAeeekkdddddddkkeee 16 1011 1027 974 AATTG 590452 206 222 TCCTTCTGCTCGeeekkdddddddkkeee 14 n/a n/a 975 AAATT 590453 207 223 TTCCTTCTGCTCGeeekkdddddddkkeee 13 n/a n/a 976 AAAT 590454 208 224 TTTCCTTCTGCTCeeekkdddddddkkeee 6 n/a n/a 977 GAAA 590455 209 225 CTTTCCTTCTGCTeeekkdddddddkkeee 0 n/a n/a 978 CGAA 590456 210 226 ACTTTCCTTCTGCeeekkdddddddkkeee 0 n/a n/a 979 TCGA 590457 211 227 TACTTTCCTTCTGeeekkdddddddkkeee n.d. n/a n/a 980 CTCG 590458 212 228 TTACTTTCCTTCTeeekkdddddddkkeee n.d. n/a n/a 981 GCTC 590459 213 229 ATTACTTTCCTTCeeekkdddddddkkeee n.d. n/a n/a 982 TGCT 590461 214 230 CATTACTTTCCTTeeekkdddddddkkeee n.d. n/a n/a 983 CTGC 590462 215 231 CCATTACTTTCCTeeekkdddddddkkeee n.d. n/a n/a 984 TCTG 590463 216 232 TCCATTACTTTCCeeekkdddddddkkeee n.d. n/a n/a 985 TTCT 590464 217 233 GTCCATTACTTTCeeekkdddddddkkeee n.d. n/a n/a 986 CTTC 590465 218 234 GGTCCATTACTTeeekkdddddddkkeee n.d. 4972 4988 987 TCCTT 590466 219 235 TGGTCCATTACTeeekkdddddddkkeee 5 4973 4989 988 TTCCT 590467 220 236 CTGGTCCATTACeeekkdddddddkkeee 11 4974 4990 989 TTTCC 590468 221 237 ACTGGTCCATTAeeekkdddddddkkeee 14 4975 4991 990 CTTTC 590469 222 238 CACTGGTCCATTeeekkdddddddkkeee 12 4976 4992 991 ACTTT 590470 223 239 TCACTGGTCCATeeekkdddddddkkeee 15 4977 4993 992 TACTT 590471 224 240 TTCACTGGTCCAeeekkdddddddkkeee 14 4978 4994 993 TTACT 590472 225 241 CTTCACTGGTCCeeekkdddddddkkeee 11 4979 4995 994 ATTAC 590473 226 242 CCTTCACTGGTCeeekkdddddddkkeee 8 4980 4996 995 CATTA 590474 227 243 ACCTTCACTGGTeeekkdddddddkkeee 44 4981 4997 996 CCATT 590475 228 244 CACCTTCACTGGeeekkdddddddkkeee 53 4982 4998 997 TCCAT 590476 229 245 ACACCTTCACTGeeekkdddddddkkeee 20 4983 4999 998 GTCCA 590477 230 246 CACACCTTCACTeeekkdddddddkkeee 12 4984 5000 999 GGTCC 590478 231 247 CCACACCTTCACeeekkdddddddkkeee 36 4985 5001 1000 TGGTC 590479 232 248 CCCACACCTTCAeeekkdddddddkkeee 18 4986 5002 1001 CTGGT 590480 233 249 CCCCACACCTTCeeekkdddddddkkeee 14 4987 5003 1002 ACTGG 590481 235 251 TTCCCCACACCTeeekkdddddddkkeee 8 4989 5005 1003 TCACT 590482 236 252 CTTCCCCACACCeeekkdddddddkkeee 29 4990 5006 1004 TTCAC 590483 237 253 GCTTCCCCACACeeekkdddddddkkeee 36 4991 5007 1005 CTTCA 590484 238 254 TGCTTCCCCACAeeekkdddddddkkeee 43 4992 5008 1006 CCTTC 590485 239 255 ATGCTTCCCCACeeekkdddddddkkeee 41 4993 5009 1007 ACCTT 590486 240 256 AATGCTTCCCCAeeekkdddddddkkeee 35 4994 5010 1008 CACCT 590487 241 257 TAATGCTTCCCCeeekkdddddddkkeee 52 4995 5011 1009 ACACC 590488 264 280 ATGCAGGCCTTCeeekkdddddddkkeee 37 5018 5034 1010 AGTCA 590489 265 281 CATGCAGGCCTTeeekkdddddddkkeee 41 5019 5035 1011 CAGTC 590490 266 282 CCATGCAGGCCTeeekkdddddddkkeee 21 5020 5036 1012 TCAGT 590491 267 283 TCCATGCAGGCCeeekkdddddddkkeee 18 5021 5037 1013 TTCAG 590492 268 284 ATCCATGCAGGCeeekkdddddddkkeee 27 5022 5038 1014 CTTCA 590493 269 285 AATCCATGCAGGeeekkdddddddkkeee 13 5023 5039 1015 CCTTC 590494 270 286 GAATCCATGCAGeeekkdddddddkkeee 9 5024 5040 1016 GCCTT 590495 271 287 GGAATCCATGCAeeekkdddddddkkeee 7 5025 5041 1017 GGCCT 590496 272 288 TGGAATCCATGCeeekkdddddddkkeee 12 5026 5042 1018 AGGCC 590497 273 289 ATGGAATCCATGeeekkdddddddkkeee 9 5027 5043 1019 CAGGC 590498 274 290 CATGGAATCCATeeekkdddddddkkeee 14 5028 5044 1020 GCAGG 590499 275 291 ACATGGAATCCAeeekkdddddddkkeee 0 5029 5045 1021 TGCAG 590500 276 292 AACATGGAATCCeeekkdddddddkkeee 10 5030 5046 1022 ATGCA 590501 277 293 GAACATGGAATCeeekkdddddddkkeee 9 5031 5047 1023 CATGC 590502 278 294 TGAACATGGAATeeekkdddddddkkeee 2 5032 5048 1024 CCATG 590503 279 295 ATGAACATGGAAeeekkdddddddkkeee 8 5033 5049 1025 TCCAT 590504 316 332 CTGCACTGGTACeeekkdddddddkkeee 3 7632 7648 1026 AGCCT 590505 317 333 CCTGCACTGGTAeeekkdddddddkkeee 17 7633 7649 1027 CAGCC 590506 318 334 ACCTGCACTGGTeeekkdddddddkkeee 12 7634 7650 1028 ACAGC 590507 319 335 GACCTGCACTGGeeekkdddddddkkeee 7 7635 7651 1029 TACAG 590508 320 336 GGACCTGCACTGeeekkdddddddkkeee 7 7636 7652 1030 GTACA 590509 321 337 AGGACCTGCACTeeekkdddddddkkeee 4 7637 7653 1031 GGTAC 590510 322 338 GAGGACCTGCACeeekkdddddddkkeee 17 7638 7654 1032 TGGTA 590511 323 339 TGAGGACCTGCAeeekkdddddddkkeee 8 7639 7655 1033 CTGGT

TABLE 14 Percent inhibition of SOD-1 mRNA by deoxy, MOE and cEt gapmerstargeting SEQ ID NO: 1 and/or 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1NO: 2 NO: 2 SEQ ISIS Start Stop % Start Stop ID NO Site Site SequenceChemistry inhibition Site Site NO 590512 169 185 CGTCGCCCTTCAeeekkdddddddkkeee 45 975 991 968 GCACG 590513 324 340 GTGAGGACCTGeeekkdddddddkkeee 21 7640 7656 1034 CACTGG 590514 325 341 AGTGAGGACCTeeekkdddddddkkeee 21 7641 7657 1035 GCACTG 590515 326 342 AAGTGAGGACCeeekkdddddddkkeee 16 7642 7658 1036 TGCACT 590516 327 343 AAAGTGAGGACeeekkdddddddkkeee 20 7643 7659 1037 CTGCAC 590517 328 344 TAAAGTGAGGAeeekkdddddddkkeee 19 7644 7660 1038 CCTGCA 590518 329 345 TTAAAGTGAGGeeekkdddddddkkeee 14 7645 7661 1039 ACCTGC 590519 330 346 ATTAAAGTGAGeeekkdddddddkkeee 51 7646 7662 1040 GACCTG 590520 331 347 GATTAAAGTGAeeekkdddddddkkeee 8 7647 7663 1041 GGACCT 590521 332 348 GGATTAAAGTGeeekkdddddddkkeee 30 7648 7664 1042 AGGACC 590522 333 349 AGGATTAAAGTeeekkdddddddkkeee 23 7649 7665 1043 GAGGAC 590523 334 350 GAGGATTAAAGeeekkdddddddkkeee 40 7650 7666 1044 TGAGGA 590524 335 351 AGAGGATTAAAeeekkdddddddkkeee 16 7651 7667 1045 GTGAGG 590525 336 352 TAGAGGATTAAeeekkdddddddkkeee 21 7652 7668 1046 AGTGAG 590526 337 353 ATAGAGGATTAeeekkdddddddkkeee 9 7653 7669 1047 AAGTGA 590527 338 354 GATAGAGGATTeeekkdddddddkkeee 8 7654 7670 1048 AAAGTG 590528 339 355 GGATAGAGGATeeekkdddddddkkeee 14 7655 7671 1049 TAAAGT 590530 340 356 TGGATAGAGGAeeekkdddddddkkeee 23 7656 7672 1050 TTAAAG 590531 341 357 CTGGATAGAGGeeekkdddddddkkeee 26 7657 7673 1051 ATTAAA 590532 342 358 TCTGGATAGAGeeekkdddddddkkeee 25 7658 7674 1052 GATTAA 590533 360 376 CTTTGGCCCACCeeekkdddddddkkeee 41 7676 7692 1053 GTGTT 590534 361 377 CCTTTGGCCCACeeekkdddddddkkeee 46 7677 7693 1054 CGTGT 590535 362 378 TCCTTTGGCCCAeeekkdddddddkkeee 39 7678 7694 1055 CCGTG 590536 363 379 ATCCTTTGGCCCeeekkdddddddkkeee n.d. 7679 7695 1056 ACCGT 590537 364 380 CATCCTTTGGCCeeekkdddddddkkeee n.d. 7680 7696 1057 CACCG 590538 365 381 TCATCCTTTGGCeeekkdddddddkkeee n.d. 7681 7697 1058 CCACC 590539 366 382 TTCATCCTTTGGeeekkdddddddkkeee n.d. 7682 7698 1059 CCCAC 590540 367 383 CTTCATCCTTTGeeekkdddddddkkeee n.d. 7683 7699 1060 GCCCA 590541 368 384 TCTTCATCCTTTeeekkdddddddkkeee n.d. 7684 7700 1061 GGCCC 590542 369 385 CTCTTCATCCTTeeekkdddddddkkeee n.d. 7685 7701 1062 TGGCC 590543 370 386 TCTCTTCATCCTeeekkdddddddkkeee n.d. 7686 7702 1063 TTGGC 590544 371 387 CTCTCTTCATCCeeekkdddddddkkeee 2 7687 7703 1064 TTTGG 590545 374 390 TGCCTCTCTTCAeeekkdddddddkkeee 6 n/a n/a 1065 TCCTT 590546 375 391 ATGCCTCTCTTCeeekkdddddddkkeee 0 n/a n/a 1066 ATCCT 590547 376 392 CATGCCTCTCTTeeekkdddddddkkeee 14 n/a n/a 1067 CATCC 590548 377 393 ACATGCCTCTCTeeekkdddddddkkeee 0 n/a n/a 1068 TCATC 590549 378 394 AACATGCCTCTCeeekkdddddddkkeee 13 n/a n/a 1069 TTCAT 590550 379 395 CAACATGCCTCTeeekkdddddddkkeee 3 n/a n/a 1070 CTTCA 590551 380 396 CCAACATGCCTCeeekkdddddddkkeee 0 n/a n/a 1071 TCTTC 590552 381 397 TCCAACATGCCTeeekkdddddddkkeee 0 n/a n/a 1072 CTCTT 590553 382 398 CTCCAACATGCCeeekkdddddddkkeee 5 n/a n/a 1073 TCTCT 590554 383 399 TCTCCAACATGCeeekkdddddddkkeee 10 n/a n/a 1074 CTCTC 590555 384 400 GTCTCCAACATGeeekkdddddddkkeee 8 n/a n/a 1075 CCTCT 590556 402 418 AGCAGTCACATTeeekkdddddddkkeee 18 8457 8473 1076 GCCCA 590557 403 419 CAGCAGTCACAeeekkdddddddkkeee 7 8458 8474 1077 TTGCCC 590558 429 445 AGACACATCGGeeekkdddddddkkeee 21 8484 8500 1078 CCACAC 590559 436 452 CTTCAATAGACAeeekkdddddddkkeee 9 8491 8507 1079 CATCG 590560 449 465 GAGATCACAGAeeekkdddddddkkeee 13 8504 8520 1080 ATCTTC 590561 501 517 TTTTTCATGGACeeekkdddddddkkeee 76 n/a n/a 1081 CACCA 590562 502 518 CTTTTTCATGGAeeekkdddddddkkeee 87 n/a n/a 1082 CCACC 590563 503 519 GCTTTTTCATGGeeekkdddddddkkeee 71 n/a n/a 1083 ACCAC 590564 504 520 TGCTTTTTCATGeeekkdddddddkkeee 51 n/a n/a 1084 GACCA 590565 505 521 CTGCTTTTTCATeeekkdddddddkkeee 65 9655 9671 1085 GGACC 590566 506 522 TCTGCTTTTTCAeeekkdddddddkkeee 55 9656 9672 1086 TGGAC 590567 507 523 ATCTGCTTTTTCeeekkdddddddkkeee 42 9657 9673 1087 ATGGA 590568 508 524 CATCTGCTTTTTeeekkdddddddkkeee 70 9658 9674 1088 CATGG 590569 509 525 TCATCTGCTTTTeeekkdddddddkkeee 71 9659 9675 1089 TCATG 590570 510 526 GTCATCTGCTTTeeekkdddddddkkeee 74 9660 9676 1090 TTCAT 590571 511 527 AGTCATCTGCTTeeekkdddddddkkeee 76 9661 9677 1091 TTTCA 590572 512 528 AAGTCATCTGCTeeekkdddddddkkeee 83 9662 9678 1092 TTTTC 590573 513 529 CAAGTCATCTGCeeekkdddddddkkeee 42 9663 9679 1093 TTTTT 590574 514 530 CCAAGTCATCTGeeekkdddddddkkeee 50 9664 9680 1094 CTTTT 590575 515 531 CCCAAGTCATCTeeekkdddddddkkeee 72 9665 9681 1095 GCTTT 590576 516 532 GCCCAAGTCATCeeekkdddddddkkeee 93 9666 9682 1096 TGCTT 590577 517 533 TGCCCAAGTCATeeekkdddddddkkeee 90 9667 9683 1097 CTGCT 590578 518 534 TTGCCCAAGTCAeeekkdddddddkkeee 92 9668 9684 1098 TCTGC 590579 524 540 CCACCTTTGCCCeeekkdddddddkkeee 91 9674 9690 1099 AAGTC 590580 525 541 TCCACCTTTGCCeeekkdddddddkkeee 88 9675 9691 1100 CAAGT 590581 526 542 TTCCACCTTTGCeeekkdddddddkkeee 87 9676 9692 1101 CCAAG 590582 527 543 TTTCCACCTTTGeeekkdddddddkkeee 78 9677 9693 1102 CCCAA 590583 528 544 ATTTCCACCTTTeeekkdddddddkkeee 63 9678 9694 1103 GCCCA 590584 529 545 CATTTCCACCTTeeekkdddddddkkeee 73 9679 9695 1104 TGCCC 590585 530 546 TCATTTCCACCTeeekkdddddddkkeee 57 9680 9696 1105 TTGCC 590586 531 547 TTCATTTCCACCeeekkdddddddkkeee 33 9681 9697 1106 TTTGC 590587 533 549 TCTTCATTTCCAeeekkdddddddkkeee 31 9683 9699 1107 CCTTT 590588 536 552 CTTTCTTCATTTeeekkdddddddkkeee 11 9686 9702 1108 CCACC 590589 537 553 ACTTTCTTCATTeeekkdddddddkkeee 15 9687 9703 1109 TCCAC 590590 538 554 TACTTTCTTCATeeekkdddddddkkeee 18 9688 9704 1110 TTCCA

TABLE 15 Percent inhibition of SOD-1 mRNA by deoxy, MOE and cEt gapmerstargeting SEQ ID NO: 1 and/or 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1NO: 2 NO: 2 SEQ ISIS Start Stop % Start Stop ID NO Site Site SequenceChemistry inhibition Site Site NO 590512 169 185 CGTCGCCCTTCAGeeekkdddddddkkeee 21 975 991 968 CACG 590591 582 598 AATTACACCACAeeekkdddddddkkeee 21 9732 9748 1111 AGCCA 590592 583 599 CAATTACACCACeeekkdddddddkkeee 33 9733 9749 1112 AAGCC 590593 584 600 CCAATTACACCAeeekkdddddddkkeee 29 9734 9750 1113 CAAGC 590594 585 601 CCCAATTACACCeeekkdddddddkkeee 29 9735 9751 1114 ACAAG 590595 588 604 GATCCCAATTACeeekkdddddddkkeee 3 9738 9754 1115 ACCAC 590596 589 605 CGATCCCAATTACeeekkdddddddkkeee 12 9739 9755 1116 ACCA 590597 590 606 GCGATCCCAATTeeekkdddddddkkeee 19 9740 9756 1117 ACACC 590598 591 607 GGCGATCCCAATeeekkdddddddkkeee 9 9741 9757 1118 TACAC 590599 592 608 GGGCGATCCCAAeeekkdddddddkkeee 18 9742 9758 1119 TTACA 590600 593 609 TGGGCGATCCCAeeekkdddddddkkeee 20 9743 9759 1120 ATTAC 590601 594 610 TTGGGCGATCCCeeekkdddddddkkeee 26 9744 9760 1121 AATTA 590602 595 611 ATTGGGCGATCCeeekkdddddddkkeee 19 9745 9761 1122 CAATT 590603 596 612 TATTGGGCGATCCeeekkdddddddkkeee 3 9746 9762 1123 CAAT 590604 597 613 TTATTGGGCGATCeeekkdddddddkkeee 15 9747 9763 1124 CCAA 590605 598 614 TTTATTGGGCGATeeekkdddddddkkeee 20 9748 9764 1125 CCCA 590606 599 615 GTTTATTGGGCGAeeekkdddddddkkeee 18 9749 9765 1126 TCCC 590607 600 616 TGTTTATTGGGCGeeekkdddddddkkeee 21 9750 9766 1127 ATCC 590608 601 617 ATGTTTATTGGGCeeekkdddddddkkeee 28 9751 9767 1128 GATC 590609 602 618 AATGTTTATTGGGeeekkdddddddkkeee 30 9752 9768 1129 CGAT 590610 603 619 GAATGTTTATTGGeeekkdddddddkkeee 14 9753 9769 1130 GCGA 590611 604 620 GGAATGTTTATTGeeekkdddddddkkeee 15 9754 9770 1131 GGCG 590612 607 623 AAGGGAATGTTTeeekkdddddddkkeee 2 9757 9773 1132 ATTGG 590613 608 624 CAAGGGAATGTTeeekkdddddddkkeee n.d. 9758 9774 1133 TATTG 590614 609 625 CCAAGGGAATGTeeekkdddddddkkeee n.d. 9759 9775 1134 TTATT 590615 610 626 TCCAAGGGAATGeeekkdddddddkkeee n.d. 9760 9776 1135 TTTAT 590616 611 627 ATCCAAGGGAATeeekkdddddddkkeee n.d. 9761 9777 1136 GTTTA 590617 612 628 CATCCAAGGGAAeeekkdddddddkkeee n.d. 9762 9778 1137 TGTTT 590618 613 629 ACATCCAAGGGAeeekkdddddddkkeee n.d. 9763 9779 1138 ATGTT 590619 614 630 TACATCCAAGGGeeekkdddddddkkeee n.d. 9764 9780 1139 AATGT 590620 615 631 CTACATCCAAGGeeekkdddddddkkeee n.d. 9765 9781 1140 GAATG 590621 616 632 ACTACATCCAAGeeekkdddddddkkeee 7 9766 9782 1141 GGAAT 590622 617 633 GACTACATCCAAeeekkdddddddkkeee 19 9767 9783 1142 GGGAA 590623 618 634 AGACTACATCCAeeekkdddddddkkeee 39 9768 9784 1143 AGGGA 590624 619 635 CAGACTACATCCeeekkdddddddkkeee 53 9769 9785 1144 AAGGG 590625 620 636 TCAGACTACATCCeeekkdddddddkkeee 57 9770 9786 1145 AAGG 590626 621 637 CTCAGACTACATCeeekkdddddddkkeee 76 9771 9787 1146 CAAG 590627 622 638 CCTCAGACTACATeeekkdddddddkkeee 58 9772 9788 1147 CCAA 590628 623 639 GCCTCAGACTACeeekkdddddddkkeee 43 9773 9789 1148 ATCCA 590629 624 640 GGCCTCAGACTAeeekkdddddddkkeee 24 9774 9790 1149 CATCC 590630 625 641 GGGCCTCAGACTeeekkdddddddkkeee 24 9775 9791 1150 ACATC 590631 643 659 GATAACAGATGAeeekkdddddddkkeee 11 9793 9809 1151 GTTAA 590632 644 660 GGATAACAGATGeeekkdddddddkkeee 32 9794 9810 1152 AGTTA 590633 645 661 AGGATAACAGATeeekkdddddddkkeee 45 9795 9811 1153 GAGTT 590634 646 662 CAGGATAACAGAeeekkdddddddkkeee 65 9796 9812 1154 TGAGT 590635 647 663 GCAGGATAACAGeeekkdddddddkkeee 58 9797 9813 1155 ATGAG 590636 648 664 AGCAGGATAACAeeekkdddddddkkeee 45 9798 9814 1156 GATGA 590637 649 665 TAGCAGGATAACeeekkdddddddkkeee 34 9799 9815 1157 AGATG 590638 650 666 CTAGCAGGATAAeeekkdddddddkkeee 39 9800 9816 1158 CAGAT 590639 651 667 GCTAGCAGGATAeeekkdddddddkkeee 10 9801 9817 1159 ACAGA 590640 652 668 AGCTAGCAGGATeeekkdddddddkkeee 15 9802 9818 1160 AACAG 590641 653 669 CAGCTAGCAGGAeeekkdddddddkkeee 21 9803 9819 1161 TAACA 590642 654 670 ACAGCTAGCAGGeeekkdddddddkkeee 20 9804 9820 1162 ATAAC 590643 655 671 TACAGCTAGCAGeeekkdddddddkkeee 40 9805 9821 1163 GATAA 590644 656 672 CTACAGCTAGCAeeekkdddddddkkeee 55 9806 9822 1164 GGATA 590645 657 673 TCTACAGCTAGCeeekkdddddddkkeee 51 9807 9823 1165 AGGAT 590646 658 674 TTCTACAGCTAGCeeekkdddddddkkeee 31 9808 9824 1166 AGGA 590647 659 675 TTTCTACAGCTAGeeekkdddddddkkeee 38 9809 9825 1167 CAGG 590648 660 676 ATTTCTACAGCTAeeekkdddddddkkeee 45 9810 9826 1168 GCAG 590649 661 677 CATTTCTACAGCTeeekkdddddddkkeee 34 9811 9827 1169 AGCA 590650 664 680 ATACATTTCTACAeeekkdddddddkkeee 57 9814 9830 1170 GCTA 590651 665 681 GATACATTTCTACeeekkdddddddkkeee 40 9815 9831 1171 AGCT 590652 683 699 GTGTTTAATGTTTeeekkdddddddkkeee 37 9833 9849 1172 ATCA 590653 684 700 AGTGTTTAATGTTeeekkdddddddkkeee 67 9834 9850 1173 TATC 590654 685 701 CAGTGTTTAATGTeeekkdddddddkkeee 54 9835 9851 1174 TTAT 590655 686 702 ACAGTGTTTAATGeeekkdddddddkkeee 56 9836 9852 1175 TTTA 590656 687 703 TACAGTGTTTAATeeekkdddddddkkeee 30 9837 9853 1176 GTTT 590657 688 704 TTACAGTGTTTAAeeekkdddddddkkeee 18 9838 9854 1177 TGTT 590658 689 705 ATTACAGTGTTTAeeekkdddddddkkeee 24 9839 9855 1178 ATGT 590659 690 706 GATTACAGTGTTTeeekkdddddddkkeee 10 9840 9856 1179 AATG 590660 691 707 AGATTACAGTGTTeeekkdddddddkkeee 45 9841 9857 1180 TAAT 590661 692 708 AAGATTACAGTGeeekkdddddddkkeee 34 9842 9858 1181 TTTAA 590662 693 709 TAAGATTACAGTeeekkdddddddkkeee 54 9843 9859 1182 GTTTA 590663 694 710 TTAAGATTACAGTeeekkdddddddkkeee 54 9844 9860 1183 GTTT 590664 772 788 ATCTTCCAAGTGAeeekkdddddddkkeee 7 9922 9938 1184 TCAT 590665 773 789 AATCTTCCAAGTGeeekkdddddddkkeee 23 9923 9939 1185 ATCA 590666 774 790 AAATCTTCCAAGTeeekkdddddddkkeee 4 9924 9940 1186 GATC 590667 775 791 CAAATCTTCCAAeeekkdddddddkkeee 18 9925 9941 1187 GTGAT

TABLE 16 Percent inhibition of SOD-1 mRNA by deoxy, MOE and cEt gapmerstargeting SEQ ID NO: 1 and/or 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1NO: 2 NO: 2 SEQ ISIS Start Stop % Start Stop ID NO Site Site SequenceChemistry inhibition Site Site NO 590512 169 185 CGTCGCCCTTeeekkdddddddkkeee 16 975 991 968 CAGCACG 590668 777 793 TACAAATCTTeeekkdddddddkkeee 17 9927 9943 1188 CCAAGTG 590669 778 794 ATACAAATCeeekkdddddddkkeee 15 9928 9944 1189 TTCCAAGT 590670 779 795 TATACAAATeeekkdddddddkkeee 11 9929 9945 1190 CTTCCAAG 590671 780 796 CTATACAAAeeekkdddddddkkeee 13 9930 9946 1191 TCTTCCAA 590672 781 797 ACTATACAAeeekkdddddddkkeee 10 9931 9947 1192 ATCTTCCA 590673 782 798 AACTATACAeeekkdddddddkkeee 28 9932 9948 1193 AATCTTCC 590674 783 799 AAACTATACeeekkdddddddkkeee 26 9933 9949 1194 AAATCTTC 590675 786 802 ATAAAACTAeeekkdddddddkkeee 14 9936 9952 1195 TACAAATC 590676 791 807 GTTTTATAAAeeekkdddddddkkeee 22 9941 9957 1196 ACTATAC 590677 793 809 GAGTTTTATAeeekkdddddddkkeee 6 9943 9959 1197 AAACTAT 590678 814 830 ATTGAAACAeeekkdddddddkkeee 22 9964 9980 1198 GACATTTT 590679 815 831 CATTGAAACeeekkdddddddkkeee 11 9965 9981 1199 AGACATTT 590680 816 832 TCATTGAAAeeekkdddddddkkeee 10 9966 9982 1200 CAGACATT 590681 817 833 GTCATTGAAeeekkdddddddkkeee 23 9967 9983 1201 ACAGACAT 590682 818 834 GGTCATTGAeeekkdddddddkkeee 11 9968 9984 1202 AACAGACA 590683 819 835 AGGTCATTGeeekkdddddddkkeee 21 9969 9985 1203 AAACAGAC 590684 820 836 CAGGTCATTeeekkdddddddkkeee 14 9970 9986 1204 GAAACAGA 590685 821 837 ACAGGTCATeeekkdddddddkkeee 14 9971 9987 1205 TGAAACAG 590686 822 838 TACAGGTCAeeekkdddddddkkeee 9 9972 9988 1206 TTGAAACA 590687 823 839 ATACAGGTCeeekkdddddddkkeee 14 9973 9989 1207 ATTGAAAC 590688 824 840 AATACAGGTeeekkdddddddkkeee 6 9974 9990 1208 CATTGAAA 590689 825 841 AAATACAGGeeekkdddddddkkeee 2 9975 9991 1209 TCATTGAA 590690 826 842 AAAATACAGeeekkdddddddkkeee n.d. 9976 9992 1210 GTCATTGA 590691 827 843 CAAAATACAeeekkdddddddkkeee n.d. 9977 9993 1211 GGTCATTG 590692 828 844 GCAAAATACeeekkdddddddkkeee n.d. 9978 9994 1212 AGGTCATT 590693 829 845 GGCAAAATAeeekkdddddddkkeee n.d. 9979 9995 1213 CAGGTCAT 590694 830 846 TGGCAAAATeeekkdddddddkkeee n.d. 9980 9996 1214 ACAGGTCA 590695 831 847 CTGGCAAAAeeekkdddddddkkeee n.d. 9981 9997 1215 TACAGGTC 590696 832 848 TCTGGCAAAeeekkdddddddkkeee n.d. 9982 9998 1216 ATACAGGT 590697 833 849 GTCTGGCAAeeekkdddddddkkeee n.d. 9983 9999 1217 AATACAGG 590698 834 850 AGTCTGGCAeeekkdddddddkkeee 1 9984 10000 1218 AAATACAG 590699 835 851 AAGTCTGGCeeekkdddddddkkeee 10 9985 10001 1219 AAAATACA 590700 836 852 TAAGTCTGGeeekkdddddddkkeee 4 9986 10002 1220 CAAAATAC 590701 837 853 TTAAGTCTGGeeekkdddddddkkeee 2 9987 10003 1221 CAAAATA 590702 853 869 AATACCCATeeekkdddddddkkeee 7 10003 10019 1222 CTGTGATT 590703 854 870 TAATACCCATeeekkdddddddkkeee 4 10004 10020 1223 CTGTGAT 590704 855 871 TTAATACCCAeeekkdddddddkkeee 2 10005 10021 1224 TCTGTGA 590705 856 872 TTTAATACCCeeekkdddddddkkeee 0 10006 10022 1225 ATCTGTG 590706 857 873 GTTTAATACCeeekkdddddddkkeee 17 10007 10023 1226 CATCTGT 590707 858 874 AGTTTAATACeeekkdddddddkkeee 10 10008 10024 1227 CCATCTG 590708 859 875 AAGTTTAATeeekkdddddddkkeee 12 10009 10025 1228 ACCCATCT 590709 860 876 CAAGTTTAATeeekkdddddddkkeee 37 10010 10026 1229 ACCCATC 590710 861 877 ACAAGTTTAeeekkdddddddkkeee 23 10011 10027 1230 ATACCCAT 590711 862 878 GACAAGTTTeeekkdddddddkkeee 24 10012 10028 1231 AATACCCA 590712 863 879 TGACAAGTTTeeekkdddddddkkeee 27 10013 10029 1232 AATACCC 590713 864 880 CTGACAAGTeeekkdddddddkkeee 10 10014 10030 1233 TTAATACC 590714 865 881 TCTGACAAGeeekkdddddddkkeee 0 10015 10031 1234 TTTAATAC 590715 866 882 TTCTGACAAeeekkdddddddkkeee 6 10016 10032 1235 GTTTAATA 590716 867 883 ATTCTGACAeeekkdddddddkkeee 9 10017 10033 1236 AGTTTAAT 590717 868 884 AATTCTGACeeekkdddddddkkeee 15 10018 10034 1237 AAGTTTAA 590718 869 885 AAATTCTGAeeekkdddddddkkeee 21 10019 10035 1238 CAAGTTTA 590719 870 886 GAAATTCTGeeekkdddddddkkeee 14 10020 10036 1239 ACAAGTTT 590720 871 887 AGAAATTCTeeekkdddddddkkeee 8 10021 10037 1240 GACAAGTT 590721 872 888 AAGAAATTCeeekkdddddddkkeee 18 10022 10038 1241 TGACAAGT 590722 891 907 TTCACAGGCTeeekkdddddddkkeee 9 10041 10057 1242 TGAATGA 590723 892 908 ATTCACAGGeeekkdddddddkkeee 11 10042 10058 1243 CTTGAATG 590724 893 909 TATTCACAGeeekkdddddddkkeee 0 10043 10059 1244 GCTTGAAT 590725 894 910 TTATTCACAGeeekkdddddddkkeee 10 10044 10060 1245 GCTTGAA 590726 895 911 TTTATTCACAeeekkdddddddkkeee 29 10045 10061 1246 GGCTTGA 590727 896 912 TTTTATTCACeeekkdddddddkkeee 28 10046 10062 1247 AGGCTTG 590728 897 913 TTTTTATTCAeeekkdddddddkkeee 31 10047 10063 1248 CAGGCTT 590729 898 914 GTTTTTATTCeeekkdddddddkkeee 10 10048 10064 1249 ACAGGCT 590731 899 915 GGTTTTTATTeeekkdddddddkkeee 22 10049 10065 1250 CACAGGC 590732 900 916 GGGTTTTTATeeekkdddddddkkeee 17 10050 10066 1251 TCACAGG 590733 901 917 AGGGTTTTTAeeekkdddddddkkeee 24 10051 10067 1252 TTCACAG 590734 902 918 CAGGGTTTTTeeekkdddddddkkeee 17 10052 10068 1253 ATTCACA 590735 903 919 ACAGGGTTTTeeekkdddddddkkeee 10 10053 10069 1254 TATTCAC 590736 904 920 TACAGGGTTTeeekkdddddddkkeee 11 10054 10070 1255 TTATTCA 590737 905 921 ATACAGGGTeeekkdddddddkkeee 3 10055 10071 1256 TTTTATTC 590738 906 922 CATACAGGGeeekkdddddddkkeee 0 10056 10072 1257 TTTTTATT 590739 907 923 CCATACAGGeeekkdddddddkkeee 1 10057 10073 1258 GTTTTTAT 590740 908 924 GCCATACAGeeekkdddddddkkeee 11 10058 10074 1259 GGTTTTTA 590741 909 925 TGCCATACAeeekkdddddddkkeee 9 10059 10075 1260 GGGTTTTT 590742 910 926 GTGCCATACeeekkdddddddkkeee 7 10060 10076 1261 AGGGTTTT 590743 911 927 AGTGCCATAeeekkdddddddkkeee 9 10061 10077 1262 CAGGGTTT 590744 938 954 TTGGATTCTTeeekkdddddddkkeee 8 10088 10104 1263 TTAATAG 590745 951 967 TTTTAGTTTGeeekkdddddddkkeee 12 n/a n/a 1264 AATTTGG

TABLE 17 Percent inhibition of SOD-1 mRNA by deoxy, MOE and cEt gapmerstargeting SEQ ID NO: 1 and/or 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1NO: 2 NO: 2 SEQ ISIS Start Stop % Start Stop ID NO Site Site SequenceChemistry inhibition Site Site NO 333611 167 186 CCGTCGCCCTeeeeeddddddddddeeeee 66 973 992 21 TCAGCACGCA 590067 202 221 CCTTCTGCTCGeeeeeddddddddddeeeee 53 1008 1027 120 AAATTGATG 590074 209 228TTACTTTCCTT eeeeeddddddddddeeeee 30 n/a n/a 127 CTGCTCGAA 590457 211 227TACTTTCCTTC eeekkdddddddkkeee 14 n/a n/a 980 TGCTCG 590458 212 228TTACTTTCCTT eeekkdddddddkkeee 22 n/a n/a 981 CTGCTC 590459 213 229ATTACTTTCCT eeekkdddddddkkeee 15 n/a n/a 982 TCTGCT 590461 214 230CATTACTTTCC eeekkdddddddkkeee 28 n/a n/a 983 TTCTGC 590462 215 231CCATTACTTTC eeekkdddddddkkeee 37 n/a n/a 984 CTTCTG 590463 216 232TCCATTACTTT eeekkdddddddkkeee 18 n/a n/a 985 CCTTCT 590082 217 236CTGGTCCATT eeeeeddddddddddeeeee 50 n/a n/a 135 ACTTTCCTTC 590464 217 233GTCCATTACTT eeekkdddddddkkeee 33 n/a n/a 986 TCCTTC 590465 218 234GGTCCATTAC eeekkdddddddkkeee 18 4972 4988 987 TTTCCTT 590130 363 382TTCATCCTTTG eeeeeddddddddddeeeee 30 7679 7698 194 GCCCACCGT 590536 363379 ATCCTTTGGC eeekkdddddddkkeee 51 7679 7695 1056 CCACCGT 590537 364380 CATCCTTTGG eeekkdddddddkkeee 38 7680 7696 1057 CCCACCG 590538 365381 TCATCCTTTGG eeekkdddddddkkeee 27 7681 7697 1058 CCCACC 590539 366382 TTCATCCTTTG eeekkdddddddkkeee 26 7682 7698 1059 GCCCAC 590540 367383 CTTCATCCTTT eeekkdddddddkkeee 35 7683 7699 1060 GGCCCA 590541 368384 TCTTCATCCTT eeekkdddddddkkeee 15 7684 7700 1061 TGGCCC 590542 369385 CTCTTCATCCT eeekkdddddddkkeee 26 7685 7701 1062 TTGGCC 590543 370386 TCTCTTCATCC eeekkdddddddkkeee 14 7686 7702 1063 TTTGGC 590138 371390 TGCCTCTCTTC eeeeeddddddddddeeeee 32 n/a n/a 202 ATCCTTTGG 590146 505524 CATCTGCTTTT eeeeeddddddddddeeeee 46 9655 9674 211 TCATGGACC 590613608 624 CAAGGGAATG eeekkdddddddkkeee 19 9758 9774 1133 TTTATTG 590614609 625 CCAAGGGAAT eeekkdddddddkkeee 36 9759 9775 1134 GTTTATT 590615610 626 TCCAAGGGAA eeekkdddddddkkeee 32 9760 9776 1135 TGTTTAT 590616611 627 ATCCAAGGGA eeekkdddddddkkeee 42 9761 9777 1136 ATGTTTA 590617612 628 CATCCAAGGG eeekkdddddddkkeee 16 9762 9778 1137 AATGTTT 590618613 629 ACATCCAAGG eeekkdddddddkkeee 30 9763 9779 1138 GAATGTT 590619614 630 TACATCCAAG eeekkdddddddkkeee 30 9764 9780 1139 GGAATGT 590620615 631 CTACATCCAA eeekkdddddddkkeee 40 9765 9781 1140 GGGAATG 590690826 842 AAAATACAGG eeekkdddddddkkeee 28 9976 9992 1210 TCATTGA 590691827 843 CAAAATACAG eeekkdddddddkkeee 23 9977 9993 1211 GTCATTG 590692828 844 GCAAAATACA eeekkdddddddkkeee 43 9978 9994 1212 GGTCATT 590693829 845 GGCAAAATAC eeekkdddddddkkeee 39 9979 9995 1213 AGGTCAT 590694830 846 TGGCAAAATA eeekkdddddddkkeee 26 9980 9996 1214 CAGGTCA 590695831 847 CTGGCAAAAT eeekkdddddddkkeee 18 9981 9997 1215 ACAGGTC 590696832 848 TCTGGCAAAA eeekkdddddddkkeee 0 9982 9998 1216 TACAGGT 590697 833849 GTCTGGCAAA eeekkdddddddkkeee 24 9983 9999 1217 ATACAGG

Example 4: Inhibition of Human SOD-1 in HepG2 Cells by Deoxy, MOE andcEt Gapmers

Modified oligonucleotides were designed targeting a superoxide dismutase1, soluble (SOD-1) nucleic acid and were tested for their effects onSOD-1 mRNA in vitro. ISIS 333611, a 5-10-5 MOE gapmer which waspreviously described in WO 2005/040180, was included as a benchmark.

The modified oligonucleotides were tested in a series of experimentsthat had similar culture conditions. The results for each experiment arepresented in separate tables shown below. Cultured HepG2 cells at adensity of 20,000 cells per well were transfected using electroporationwith 4,000 nM modified oligonucleotide. After a treatment period ofapproximately 24 hours, RNA was isolated from the cells and SOD-1 mRNAlevels were measured by quantitative real-time PCR.

Human primer probe set RTS3898 was used to measure mRNA levels. SOD-1mRNA levels were adjusted according to total RNA content, as measured byRIBOGREEN®. Results are presented as percent inhibition of SOD-1,relative to untreated control cells. ‘n.d.’ indicates that inhibitionlevels were not measured.

The newly designed modified oligonucleotides in the Tables below weredesigned as deoxy, MOE, and cEt gapmers or 5-10-5 gapmers. The 5-10-5MOE gapmers are 20 nucleosides in length, wherein the central gapsegment is comprised of ten 2′-deoxyribonucleosides and is flanked bywing segments on the 5′ direction and the 3′ direction comprising fivenucleosides each. Each nucleoside in the 5′ wing segment and eachnucleoside in the 3′ wing segment has a 2′-MOE modification. The deoxy,MOE and cEt oligonucleotides are 17 nucleosides in length wherein thenucleoside has a MOE sugar modification, a cEt sugar modification, or adeoxy moiety. The sugar chemistry of each oligonucleotide is denoted asin the Chemistry column, where 1′ indicates a cEt modified sugar; ‘d’indicates a 2′-deoxyribose; and ‘e’ indicates a 2′-MOE modified sugar.The internucleoside linkages throughout each gapmer are phosphorothioatelinkages. All cytosine residues throughout each gapmer are5-methylcytosines. “Start site” indicates the 5′-most nucleoside towhich the gapmer is targeted in the human gene sequence. Each gapmerlisted in the Tables below is targeted to either the human SOD-1 mRNA,designated herein as SEQ ID NO: 1 (GENBANK Accession No. NM_000454.4) orthe human SOD-1 genomic sequence, designated herein as SEQ ID NO: 2(GENBANK Accession No. NT_011512.10 truncated from nucleotides 18693000to Ser. No. 18/704,000). ‘n/a’ indicates that the modifiedoligonucleotide does not target that particular gene sequence with 100%complementarity.

TABLE 18 Percent inhibition of SOD-1 mRNA by 5-10-5 MOE gapmerstargeting SEQ ID NO: 1 and/or 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1NO: 2 NO: 2 SEQ ISIS Start Stop % Start Stop ID NO Site Site Sequenceinhibition Site Site NO 596168 3 22 CTCGCCCACTCTGGCCCCAA 45 809 828 1265596169 5 24 GCCTCGCCCACTCTGGCCCC 37 811 830 1266 596170 7 26GCGCCTCGCCCACTCTGGCC 33 813 832 1267 596171 9 28 CCGCGCCTCGCCCACTCTGG 27815 834 1268 596172 11 30 CTCCGCGCCTCGCCCACTCT 40 817 836 1269 596173 1332 ACCTCCGCGCCTCGCCCACT 77 819 838 1270 596174 15 34AGACCTCCGCGCCTCGCCCA 72 821 840 1271 596175 17 36 CCAGACCTCCGCGCCTCGCC46 823 842 1272 596176 19 38 GGCCAGACCTCCGCGCCTCG 49 825 844 1273 15050821 40 TAGGCCAGACCTCCGCGCCT 33 827 846 107 596177 23 42TATAGGCCAGACCTCCGCGC 40 829 848 1274 596178 25 44 TTTATAGGCCAGACCTCCGC69 831 850 1275 150509 27 46 ACTTTATAGGCCAGACCTCC 64 833 852 108 59617931 50 GACTACTTTATAGGCCAGAC 74 837 856 1276 596180 33 52GCGACTACTTTATAGGCCAG 19 839 858 1277 596181 37 56 CTCCGCGACTACTTTATAGG27 843 862 1278 596182 39 58 GTCTCCGCGACTACTTTATA 22 845 864 1279 59618341 60 CCGTCTCCGCGACTACTTTA 20 847 866 1280 596184 43 62CCCCGTCTCCGCGACTACTT 16 849 868 1281 596185 45 64 CACCCCGTCTCCGCGACTAC13 851 870 1282 596186 47 66 AGCACCCCGTCTCCGCGACT 24 853 872 1283 59618749 68 CCAGCACCCCGTCTCCGCGA 38 855 874 1284 596188 51 70AACCAGCACCCCGTCTCCGC 11 857 876 1285 596189 53 72 CAAACCAGCACCCCGTCTCC13 859 878 1286 596190 55 74 CGCAAACCAGCACCCCGTCT 21 861 880 1287 15051057 76 GACGCAAACCAGCACCCCGT 45 863 882 109 596191 59 78ACGACGCAAACCAGCACCCC 30 865 884 1288 596192 61 80 CTACGACGCAAACCAGCACC19 867 886 1289 596193 63 82 GACTACGACGCAAACCAGCA 40 869 888 1290 59619465 84 GAGACTACGACGCAAACCAG 23 871 890 1291 596195 67 86AGGAGACTACGACGCAAACC 35 873 892 1292 596196 69 88 GCAGGAGACTACGACGCAAA33 875 894 1293 596197 71 90 CTGCAGGAGACTACGACGCA 36 877 896 1294 59619873 92 CGCTGCAGGAGACTACGACG 23 879 898 1295 596199 91 110TGCAACGGAAACCCCAGACG 21 897 916 1296 596200 93 112 ACTGCAACGGAAACCCCAGA43 899 918 1297 596201 97 116 GAGGACTGCAACGGAAACCC 24 903 922 1298596202 99 118 CCGAGGACTGCAACGGAAAC 29 905 924 1299 596203 101 120TTCCGAGGACTGCAACGGAA 5 907 926 1300 150438 103 122 GGTTCCGAGGACTGCAACGG35 909 928 110 345716 105 124 CTGGTTCCGAGGACTGCAAC 51 911 930 1301150439 107 126 TCCTGGTTCCGAGGACTGCA 24 913 932 111 596204 109 128GGTCCTGGTTCCGAGGACTG 14 915 934 1302 150440 111 130 GAGGTCCTGGTTCCGAGGAC31 917 936 112 596205 113 132 CCGAGGTCCTGGTTCCGAGG 18 919 938 1303345718 115 134 CGCCGAGGTCCTGGTTCCGA 24 921 940 1304 596206 117 136CACGCCGAGGTCCTGGTTCC 23 923 942 1305 596207 119 138 GCCACGCCGAGGTCCTGGTT38 925 944 1306 596208 123 142 CTAGGCCACGCCGAGGTCCT 39 929 948 1307345720 125 144 CGCTAGGCCACGCCGAGGTC 52 931 950 1308 596209 127 146CTCGCTAGGCCACGCCGAGG 46 933 952 1309 596210 129 148 AACTCGCTAGGCCACGCCGA44 935 954 1310 596211 131 150 ATAACTCGCTAGGCCACGCC 12 937 956 1311596212 133 152 CCATAACTCGCTAGGCCACG 22 939 958 1312 345722 135 154CGCCATAACTCGCTAGGCCA 59 941 960 1313 150442 137 156 GTCGCCATAACTCGCTAGGC40 943 962 113 146143 157 176 TCAGCACGCACACGGCCTTC 52 963 982 114 195753159 178 CTTCAGCACGCACACGGCCT 57 965 984 115 333607 161 180CCCTTCAGCACGCACACGGC 37 967 986 116 333608 163 182 CGCCCTTCAGCACGCACACG23 969 988 117 333611 167 186 CCGTCGCCCTTCAGCACGCA 67 973 992 21 596213171 190 TGGGCCGTCGCCCTTCAGCA 12 977 996 1314 596214 173 192ACTGGGCCGTCGCCCTTCAG 26 979 998 1315 596215 175 194 GCACTGGGCCGTCGCCCTTC14 981 1000 1316 596216 177 196 CTGCACTGGGCCGTCGCCCT 24 983 1002 1317596217 181 200 TGCCCTGCACTGGGCCGTCG 38 987 1006 1318 596218 183 202GATGCCCTGCACTGGGCCGT 15 989 1008 1319 596219 185 204ATGATGCCCTGCACTGGGCC 20 991 1010 1320 596220 189 208ATTGATGATGCCCTGCACTG 8 995 1014 1321 596221 191 210 AAATTGATGATGCCCTGCAC14 997 1016 1322 596222 193 212 CGAAATTGATGATGCCCTGC 32 999 1018 1323596223 195 214 CTCGAAATTGATGATGCCCT 31 1001 1020 1324 596224 197 216TGCTCGAAATTGATGATGCC 20 1003 1022 1325 596225 199 218TCTGCTCGAAATTGATGATG 14 1005 1024 1326 596226 201 220CTTCTGCTCGAAATTGATGA 11 1007 1026 1327 596227 240 259TTTAATGCTTCCCCACACCT 15 4994 5013 1328 596228 242 261CCTTTAATGCTTCCCCACAC 1 4996 5015 1329 596229 244 263GTCCTTTAATGCTTCCCCAC 9 4998 5017 1330

TABLE 19 Percent inhibition of SOD-1 mRNA by deoxy, MOE and cEt gapmerstargeting SEQ ID NO: 1 and/or 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1NO: 2 NO: 2 SEQ ISIS Start Stop % Start Stop ID NO Site Site SequenceChemistry inhibition Site Site NO 596530 164 180 CCCTTCAGCAeekkddddddddkkeee 74 970 986 1331 CGCACAC 596721 164 180 CCCTTCAGCAeekkdddddddddkkee 81 970 986 1331 CGCACAC 596531 165 181 GCCCTTCAGCeekkddddddddkkeee 75 971 987 1332 ACGCACA 596722 165 181 GCCCTTCAGCeekkdddddddddkkee 60 971 987 1332 ACGCACA 596532 166 182 CGCCCTTCAGeekkddddddddkkeee 67 972 988 1333 CACGCAC 596723 166 182 CGCCCTTCAGeekkdddddddddkkee 73 972 988 1333 CACGCAC 333611 167 186 CCGTCGCCCTeeeeeddddddddddeeeee 73 973 992 21 TCAGCACGCA 596720 167 183 TCGCCCTTCAeekkddddddddkkeee 56 973 989 966 GCACGCA 596911 167 183 TCGCCCTTCAeekkdddddddddkkee 63 973 989 966 GCACGCA 596533 168 184 GTCGCCCTTCeekkddddddddkkeee 60 974 990 967 AGCACGC 596724 168 184 GTCGCCCTTCeekkdddddddddkkee 72 974 990 967 AGCACGC 596534 169 185 CGTCGCCCTTeekkddddddddkkeee 52 975 991 968 CAGCACG 596725 169 185 CGTCGCCCTTeekkdddddddddkkee 43 975 991 968 CAGCACG 596535 170 186 CCGTCGCCCTeekkddddddddkkeee 71 976 992 969 TCAGCAC 596726 170 186 CCGTCGCCCTeekkdddddddddkkee 75 976 992 969 TCAGCAC 596536 171 187 GCCGTCGCCCeekkddddddddkkeee 64 977 993 970 TTCAGCA 596727 171 187 GCCGTCGCCCeekkdddddddddkkee 57 977 993 970 TTCAGCA 596537 577 593 CACCACAAGCeekkddddddddkkeee 48 9727 9743 1334 CAAACGA 596728 577 593 CACCACAAGCeekkdddddddddkkee 46 9727 9743 1334 CAAACGA 596538 578 594 ACACCACAAGeekkddddddddkkeee 27 9728 9744 1335 CCAAACG 596729 578 594 ACACCACAAGeekkdddddddddkkee 45 9728 9744 1335 CCAAACG 596539 579 595 TACACCACAAeekkddddddddkkeee 56 9729 9745 1336 GCCAAAC 596730 579 595 TACACCACAAeekkdddddddddkkee 63 9729 9745 1336 GCCAAAC 596540 580 596 TTACACCACAeekkddddddddkkeee 60 9730 9746 1337 AGCCAAA 596731 580 596 TTACACCACAeekkdddddddddkkee 63 9730 9746 1337 AGCCAAA 596541 581 597 ATTACACCACeekkddddddddkkeee 46 9731 9747 1338 AAGCCAA 596732 581 597 ATTACACCACeekkdddddddddkkee 63 9731 9747 1338 AAGCCAA 596542 582 598 AATTACACCAeekkddddddddkkeee 62 9732 9748 1111 CAAGCCA 596733 582 598 AATTACACCAeekkdddddddddkkee 56 9732 9748 1111 CAAGCCA 596543 583 599 CAATTACACCeekkddddddddkkeee 58 9733 9749 1112 ACAAGCC 596734 583 599 CAATTACACCeekkdddddddddkkee 61 9733 9749 1112 ACAAGCC 596544 584 600 CCAATTACACeekkddddddddkkeee 66 9734 9750 1113 CACAAGC 596735 584 600 CCAATTACACeekkdddddddddkkee 73 9734 9750 1113 CACAAGC 596545 585 601 CCCAATTACAeekkddddddddkkeee 63 9735 9751 1114 CCACAAG 596736 585 601 CCCAATTACAeekkdddddddddkkee 74 9735 9751 1114 CCACAAG 596546 588 604 GATCCCAATTeekkddddddddkkeee 41 9738 9754 1115 ACACCAC 596737 588 604 GATCCCAATTeekkdddddddddkkee 58 9738 9754 1115 ACACCAC 596547 589 605 CGATCCCAATeekkddddddddkkeee 57 9739 9755 1116 TACACCA 596738 589 605 CGATCCCAATeekkdddddddddkkee 59 9739 9755 1116 TACACCA 596548 590 606 GCGATCCCAAeekkddddddddkkeee 31 9740 9756 1117 TTACACC 596739 590 606 GCGATCCCAAeekkdddddddddkkee 58 9740 9756 1117 TTACACC 596549 591 607 GGCGATCCCAeekkddddddddkkeee 33 9741 9757 1118 ATTACAC 596740 591 607 GGCGATCCCAeekkdddddddddkkee 66 9741 9757 1118 ATTACAC 596550 592 608 GGGCGATCCCeekkddddddddkkeee 30 9742 9758 1119 AATTACA 596741 592 608 GGGCGATCCCeekkdddddddddkkee 30 9742 9758 1119 AATTACA 596551 593 609 TGGGCGATCCeekkddddddddkkeee 19 9743 9759 1120 CAATTAC 596742 593 609 TGGGCGATCCeekkdddddddddkkee 46 9743 9759 1120 CAATTAC 596552 594 610 TTGGGCGATCeekkddddddddkkeee 14 9744 9760 1121 CCAATTA 596743 594 610 TTGGGCGATCeekkdddddddddkkee 5 9744 9760 1121 CCAATTA 596553 595 611 ATTGGGCGATeekkddddddddkkeee 2 9745 9761 1122 CCCAATT 596744 595 611 ATTGGGCGATeekkdddddddddkkee 23 9745 9761 1122 CCCAATT 596554 596 612 TATTGGGCGAeekkddddddddkkeee 19 9746 9762 1123 TCCCAAT 596745 596 612 TATTGGGCGAeekkdddddddddkkee 6 9746 9762 1123 TCCCAAT 596555 597 613 TTATTGGGCGeekkddddddddkkeee 41 9747 9763 1124 ATCCCAA 596746 597 613 TTATTGGGCGeekkdddddddddkkee 41 9747 9763 1124 ATCCCAA 596556 598 614 TTTATTGGGCeekkddddddddkkeee 34 9748 9764 1125 GATCCCA 596747 598 614 TTTATTGGGCeekkdddddddddkkee 46 9748 9764 1125 GATCCCA 596557 599 615 GTTTATTGGGeekkddddddddkkeee 54 9749 9765 1126 CGATCCC 596748 599 615 GTTTATTGGGeekkdddddddddkkee 68 9749 9765 1126 CGATCCC 596558 600 616 TGTTTATTGGeekkddddddddkkeee 50 9750 9766 1127 GCGATCC 596749 600 616 TGTTTATTGGeekkdddddddddkkee 47 9750 9766 1127 GCGATCC 596559 601 617 ATGTTTATTGeekkddddddddkkeee 76 9751 9767 1128 GGCGATC 596750 601 617 ATGTTTATTGeekkdddddddddkkee 64 9751 9767 1128 GGCGATC 596560 602 618 AATGTTTATTeekkddddddddkkeee 61 9752 9768 1129 GGGCGAT 596751 602 618 AATGTTTATTeekkdddddddddkkee 64 9752 9768 1129 GGGCGAT 596561 603 619 GAATGTTTATeekkddddddddkkeee 47 9753 9769 1130 TGGGCGA 596752 603 619 GAATGTTTATeekkdddddddddkkee 65 9753 9769 1130 TGGGCGA 596562 604 620 GGAATGTTTAeekkddddddddkkeee 37 9754 9770 1131 TTGGGCG 596753 604 620 GGAATGTTTAeekkdddddddddkkee 58 9754 9770 1131 TTGGGCG 596563 608 624 CAAGGGAATGeekkddddddddkkeee 43 9758 9774 1133 TTTATTG 596754 608 624 CAAGGGAATGeekkdddddddddkkee 38 9758 9774 1133 TTTATTG 596564 609 625 CCAAGGGAATeekkddddddddkkeee 57 9759 9775 1134 GTTTATT 596755 609 625 CCAAGGGAATeekkdddddddddkkee 52 9759 9775 1134 GTTTATT 596565 610 626 TCCAAGGGAAeekkddddddddkkeee 27 9760 9776 1135 TGTTTAT 596756 610 626 TCCAAGGGAAeekkdddddddddkkee 57 9760 9776 1135 TGTTTAT 596566 611 627 ATCCAAGGGAeekkddddddddkkeee 35 9761 9777 1136 ATGTTTA 596757 611 627 ATCCAAGGGAeekkdddddddddkkee 39 9761 9777 1136 ATGTTTA 596567 616 632 ACTACATCCAeekkddddddddkkeee 42 9766 9782 1141 AGGGAAT 596758 616 632 ACTACATCCAeekkdddddddddkkee 48 9766 9782 1141 AGGGAAT

TABLE 20 Percent inhibition of SOD-1 mRNA by deoxy, MOE and cEt gapmerstargeting SEQ ID NO: 1 and/or 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1NO: 2 NO: 2 SEQ ISIS Start Stop % Start Stop ID NO Site Site SequenceChemistry inhibition Site Site NO 333611 167 186 CCGTCGCCCTeeeeeddddddddddeeeee 64 973 992 21 TCAGCACGCA 596720 167 183 TCGCCCTTCAeekkddddddddkkeee 56 973 989 966 GCACGCA 596911 167 183 TCGCCCTTCAeekkdddddddddkkee 60 973 989 966 GCACGCA 596568 617 633 GACTACATCCeekkdddddddddkkee 50 9767 9783 1142 AAGGGAA 596759 617 633 GACTACATCCeekkdddddddddkkee 57 9767 9783 1142 AAGGGAA 596569 618 634 AGACTACATCeekkddddddddkkeee 53 9768 9784 1143 CAAGGGA 596760 618 634 AGACTACATCeekkdddddddddkkee 55 9768 9784 1143 CAAGGGA 596570 619 635 CAGACTACATeekkddddddddkkeee 81 9769 9785 1144 CCAAGGG 596761 619 635 CAGACTACATeekkdddddddddkkee 78 9769 9785 1144 CCAAGGG 596571 620 636 TCAGACTACAeekkddddddddkkeee 79 9770 9786 1145 TCCAAGG 596762 620 636 TCAGACTACAeekkdddddddddkkee 78 9770 9786 1145 TCCAAGG 596572 621 637 CTCAGACTACeekkddddddddkkeee 85 9771 9787 1146 ATCCAAG 596763 621 637 CTCAGACTACeekkdddddddddkkee 76 9771 9787 1146 ATCCAAG 596573 622 638 CCTCAGACTAeekkddddddddkkeee 73 9772 9788 1147 CATCCAA 596764 622 638 CCTCAGACTAeekkdddddddddkkee 87 9772 9788 1147 CATCCAA 596574 623 639 GCCTCAGACTeekkddddddddkkeee 69 9773 9789 1148 ACATCCA 596765 623 639 GCCTCAGACTeekkdddddddddkkee 82 9773 9789 1148 ACATCCA 596575 624 640 GGCCTCAGACeekkddddddddkkeee 70 9774 9790 1149 TACATCC 596766 624 640 GGCCTCAGACeekkdddddddddkkee 76 9774 9790 1149 TACATCC 596576 625 641 GGGCCTCAGAeekkddddddddkkeee 55 9775 9791 1150 CTACATC 596767 625 641 GGGCCTCAGAeekkdddddddddkkee 58 9775 9791 1150 CTACATC 596577 640 656 AACAGATGAeekkddddddddkkeee 73 9790 9806 1339 GTTAAGGG 596768 640 656 AACAGATGAeekkdddddddddkkee 86 9790 9806 1339 GTTAAGGG 596578 641 657 TAACAGATGAeekkddddddddkkeee 68 9791 9807 1340 GTTAAGG 596769 641 657 TAACAGATGAeekkdddddddddkkee 80 9791 9807 1340 GTTAAGG 596579 642 658 ATAACAGATGeekkddddddddkkeee 27 9792 9808 1341 AGTTAAG 596770 642 658 ATAACAGATGeekkdddddddddkkee 42 9792 9808 1341 AGTTAAG 596580 643 659 GATAACAGATeekkddddddddkkeee 41 9793 9809 1151 GAGTTAA 596771 643 659 GATAACAGATeekkdddddddddkkee 28 9793 9809 1151 GAGTTAA 596581 644 660 GGATAACAGeekkddddddddkkeee 63 9794 9810 1152 ATGAGTTA 596772 644 660 GGATAACAGeekkdddddddddkkee 63 9794 9810 1152 ATGAGTTA 596582 645 661 AGGATAACAeekkddddddddkkeee 84 9795 9811 1153 GATGAGTT 596773 645 661 AGGATAACAeekkdddddddddkkee 86 9795 9811 1153 GATGAGTT 596583 646 662 CAGGATAACeekkddddddddkkeee 95 9796 9812 1154 AGATGAGT 596774 646 662 CAGGATAACeekkdddddddddkkee 96 9796 9812 1154 AGATGAGT 596584 647 663 GCAGGATAAeekkddddddddkkeee 79 9797 9813 1155 CAGATGAG 596775 647 663 GCAGGATAAeekkdddddddddkkee 86 9797 9813 1155 CAGATGAG 596585 651 667 GCTAGCAGGeekkddddddddkkeee 19 9801 9817 1159 ATAACAGA 596776 651 667 GCTAGCAGGeekkdddddddddkkee 43 9801 9817 1159 ATAACAGA 596586 652 668 AGCTAGCAGeekkddddddddkkeee 57 9802 9818 1160 GATAACAG 596777 652 668 AGCTAGCAGeekkdddddddddkkee 54 9802 9818 1160 GATAACAG 596587 653 669 CAGCTAGCAGeekkddddddddkkeee 71 9803 9819 1161 GATAACA 596778 653 669 CAGCTAGCAGeekkdddddddddkkee 61 9803 9819 1161 GATAACA 596588 654 670 ACAGCTAGCAeekkddddddddkkeee 79 9804 9820 1162 GGATAAC 596779 654 670 ACAGCTAGCAeekkdddddddddkkee 83 9804 9820 1162 GGATAAC 596589 655 671 TACAGCTAGCeekkddddddddkkeee 85 9805 9821 1163 AGGATAA 596780 655 671 TACAGCTAGCeekkdddddddddkkee 86 9805 9821 1163 AGGATAA 596590 656 672 CTACAGCTAGeekkddddddddkkeee 87 9806 9822 1164 CAGGATA 596781 656 672 CTACAGCTAGeekkdddddddddkkee 91 9806 9822 1164 CAGGATA 596591 657 673 TCTACAGCTAeekkddddddddkkeee 75 9807 9823 1165 GCAGGAT 596782 657 673 TCTACAGCTAeekkdddddddddkkee 83 9807 9823 1165 GCAGGAT 596592 658 674 TTCTACAGCTeekkddddddddkkeee 74 9808 9824 1166 AGCAGGA 596783 658 674 TTCTACAGCTeekkdddddddddkkee 79 9808 9824 1166 AGCAGGA 596593 659 675 TTTCTACAGCeekkddddddddkkeee 76 9809 9825 1167 TAGCAGG 596784 659 675 TTTCTACAGCeekkdddddddddkkee 84 9809 9825 1167 TAGCAGG 596594 660 676 ATTTCTACAGeekkddddddddkkeee 66 9810 9826 1168 CTAGCAG 596785 660 676 ATTTCTACAGeekkdddddddddkkee 75 9810 9826 1168 CTAGCAG 596595 665 681 GATACATTTCeekkddddddddkkeee 64 9815 9831 1171 TACAGCT 596786 665 681 GATACATTTCeekkdddddddddkkee 77 9815 9831 1171 TACAGCT 596596 666 682 GGATACATTTeekkddddddddkkeee 75 9816 9832 1342 CTACAGC 596787 666 682 GGATACATTTeekkdddddddddkkee 84 9816 9832 1342 CTACAGC 596597 667 683 AGGATACATTeekkddddddddkkeee 60 9817 9833 1343 TCTACAG 596788 667 683 AGGATACATTeekkdddddddddkkee 77 9817 9833 1343 TCTACAG 596598 668 684 CAGGATACATeekkddddddddkkeee 79 9818 9834 1344 TTCTACA 596789 668 684 CAGGATACATeekkdddddddddkkee 85 9818 9834 1344 TTCTACA 596599 672 688 TTATCAGGATeekkddddddddkkeee 57 9822 9838 1345 ACATTTC 596790 672 688 TTATCAGGATeekkdddddddddkkee 67 9822 9838 1345 ACATTTC 596600 674 690 GTTTATCAGGeekkddddddddkkeee 85 9824 9840 1346 ATACATT 596791 674 690 GTTTATCAGGeekkdddddddddkkee 88 9824 9840 1346 ATACATT 596601 675 691 TGTTTATCAGeekkddddddddkkeee 70 9825 9841 1347 GATACAT 596792 675 691 TGTTTATCAGeekkdddddddddkkee 83 9825 9841 1347 GATACAT 596602 676 692 ATGTTTATCAeekkddddddddkkeee 85 9826 9842 1348 GGATACA 596793 676 692 ATGTTTATCAeekkdddddddddkkee 81 9826 9842 1348 GGATACA 596603 677 693 AATGTTTATCeekkddddddddkkeee 89 9827 9843 1349 AGGATAC 596794 677 693 AATGTTTATCeekkdddddddddkkee 90 9827 9843 1349 AGGATAC 596604 678 694 TAATGTTTATeekkddddddddkkeee 90 9828 9844 1350 CAGGATA 596795 678 694 TAATGTTTATeekkdddddddddkkee 85 9828 9844 1350 CAGGATA 596605 679 695 TTAATGTTTAeekkddddddddkkeee 90 9829 9845 1351 TCAGGAT 596796 679 695 TTAATGTTTAeekkdddddddddkkee 92 9829 9845 1351 TCAGGAT

Example 5: Inhibition of Human SOD-1 in HepG2 Cells by Deoxy, MOE andcEt Gapmers

Modified oligonucleotides were designed targeting an SOD-1 nucleic acidand were tested for their effects on SOD-1 mRNA in vitro. ISIS 333611, a5-10-5 MOE gapmer, which was previously described in WO 2005/040180, wasincluded as a benchmark.

The modified oligonucleotides were tested in a series of experimentsthat had similar culture conditions. The results for each experiment arepresented in separate tables shown below. Cultured HepG2 cells at adensity of 20,000 cells per well were transfected using electroporationwith 5,000 nM modified oligonucleotide. After a treatment period ofapproximately 24 hours, RNA was isolated from the cells and SOD-1 mRNAlevels were measured by quantitative real-time PCR.

Human primer probe set RTS3898 was used to measure mRNA levels. SOD-1mRNA levels were adjusted according to total RNA content, as measured byRIBOGREEN®. Results are presented as percent inhibition of SOD-1,relative to untreated control cells. ‘n.d.’ indicates that inhibitionlevels were not measured.

The newly designed modified oligonucleotides in the Tables below weredesigned as deoxy, MOE, and cEt gapmers. The gapmers are 17 nucleosidesin length wherein each nucleoside has a MOE sugar modification, a cEtsugar modification, or a deoxy moiety. The sugar chemistry of eacholigonucleotide is denoted as in the Chemistry column, where ‘k’indicates a cEt modified sugar; ‘d’ indicates a 2′-deoxyribose; and ‘e’indicates a 2′-MOE modified sugar. The internucleoside linkagesthroughout each gapmer are phosphorothioate linkages. All cytosineresidues throughout each gapmer are 5-methylcytosines. “Start site”indicates the 5′-most nucleoside to which the gapmer is targeted in thehuman gene sequence. Each gapmer listed in the Tables below is targetedto either the human SOD-1 mRNA, designated herein as SEQ ID NO: 1(GENBANK Accession No. NM_000454.4) or the human SOD-1 genomic sequence,designated herein as SEQ ID NO: 2 (GENBANK Accession No. NT_011512.10truncated from nucleotides 18693000 to Ser. No. 18/704,000). ‘n/a’indicates that the modified oligonucleotide does not target thatparticular gene sequence with 100% complementarity.

TABLE 21 Percent inhibition of SOD-1 mRNA by deoxy, MOE and cEt gapmerstargeting SEQ ID NO: 1 and/or 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1NO: 2 NO: 2 SEQ ISIS Start Stop % Start Stop ID NO Site Site SequenceChemistry inhibition Site Site NO 333611 167 186 CCGTCGCCCTTeeeeeddddddddddeeeee 71 973 992 21 CAGCACGCA 596720 167 183 TCGCCCTTCAGeekkddddddddkkeee 53 973 989 966 CACGCA 596911 167 183 TCGCCCTTCAGeekkdddddddddkkee 61 973 989 966 CACGCA 596606 681 697 GTTTAATGTTTeekkddddddddkkeee 87 9831 9847 1352 ATCAGG 596797 681 697 GTTTAATGTTTeekkdddddddddkkee 92 9831 9847 1352 ATCAGG 596607 683 699 GTGTTTAATGTeekkddddddddkkeee 86 9833 9849 1172 TTATCA 596798 683 699 GTGTTTAATGTeekkdddddddddkkee 86 9833 9849 1172 TTATCA 596608 684 700 AGTGTTTAATeekkddddddddkkeee 88 9834 9850 1173 GTTTATC 596799 684 700 AGTGTTTAATeekkdddddddddkkee 80 9834 9850 1173 GTTTATC 596609 685 701 CAGTGTTTAATeekkddddddddkkeee 77 9835 9851 1174 GTTTAT 596800 685 701 CAGTGTTTAATeekkdddddddddkkee 85 9835 9851 1174 GTTTAT 596610 686 702 ACAGTGTTTAeekkddddddddkkeee 83 9836 9852 1175 ATGTTTA 596801 686 702 ACAGTGTTTAeekkdddddddddkkee 84 9836 9852 1175 ATGTTTA 596611 690 706 GATTACAGTGeekkddddddddkkeee 54 9840 9856 1179 TTTAATG 596802 690 706 GATTACAGTGeekkdddddddddkkee 61 9840 9856 1179 TTTAATG 596612 691 707 AGATTACAGTeekkddddddddkkeee 68 9841 9857 1180 GTTTAAT 596803 691 707 AGATTACAGTeekkdddddddddkkee 63 9841 9857 1180 GTTTAAT 596613 697 713 CTTTTAAGATTeekkddddddddkkeee 62 9847 9863 1353 ACAGTG 596804 697 713 CTTTTAAGATTeekkdddddddddkkee 53 9847 9863 1353 ACAGTG 596614 699 715 CACTTTTAAGeekkddddddddkkeee 37 9849 9865 1354 ATTACAG 596805 699 715 CACTTTTAAGeekkdddddddddkkee 49 9849 9865 1354 ATTACAG 596615 710 726 TCACACAATTeekkddddddddkkeee 28 9860 9876 1355 ACACTTT 596806 710 726 TCACACAATTeekkdddddddddkkee 39 9860 9876 1355 ACACTTT 596616 711 727 GTCACACAATeekkddddddddkkeee 28 9861 9877 1356 TACACTT 596807 711 727 GTCACACAATeekkdddddddddkkee 35 9861 9877 1356 TACACTT 596617 713 729 AAGTCACACAeekkddddddddkkeee 41 9863 9879 1357 ATTACAC 596808 713 729 AAGTCACACAeekkdddddddddkkee 37 9863 9879 1357 ATTACAC 596618 737 753 AGGTACTTTAeekkddddddddkkeee 35 9887 9903 1358 AAGCAAC 596809 737 753 AGGTACTTTAeekkdddddddddkkee 42 9887 9903 1358 AAGCAAC 596619 739 755 ACAGGTACTTeekkddddddddkkeee 14 9889 9905 1359 TAAAGCA 596810 739 755 ACAGGTACTTeekkdddddddddkkee 20 9889 9905 1359 TAAAGCA 596620 740 756 TACAGGTACTeekkddddddddkkeee 23 9890 9906 1360 TTAAAGC 596811 740 756 TACAGGTACTeekkdddddddddkkee 26 9890 9906 1360 TTAAAGC 596621 741 757 CTACAGGTACeekkddddddddkkeee 2 9891 9907 1361 TTTAAAG 596812 741 757 CTACAGGTACeekkdddddddddkkee 16 9891 9907 1361 TTTAAAG 596622 743 759 CACTACAGGTeekkddddddddkkeee 27 9893 9909 1362 ACTTTAA 596813 743 759 CACTACAGGTeekkdddddddddkkee 38 9893 9909 1362 ACTTTAA 596623 744 760 TCACTACAGGeekkddddddddkkeee 27 9894 9910 1363 TACTTTA 596814 744 760 TCACTACAGGeekkdddddddddkkee 35 9894 9910 1363 TACTTTA 596624 745 761 CTCACTACAGeekkddddddddkkeee 40 9895 9911 1364 GTACTTT 596815 745 761 CTCACTACAGeekkdddddddddkkee 54 9895 9911 1364 GTACTTT 596625 746 762 TCTCACTACAeekkddddddddkkeee 42 9896 9912 1365 GGTACTT 596816 746 762 TCTCACTACAeekkdddddddddkkee 46 9896 9912 1365 GGTACTT 596626 747 763 TTCTCACTACAeekkddddddddkkeee 26 9897 9913 1366 GGTACT 596817 747 763 TTCTCACTACAeekkdddddddddkkee 37 9897 9913 1366 GGTACT 596627 748 764 TTTCTCACTACeekkddddddddkkeee 35 9898 9914 1367 AGGTAC 596818 748 764 TTTCTCACTACeekkdddddddddkkee 45 9898 9914 1367 AGGTAC 596628 749 765 GTTTCTCACTAeekkddddddddkkeee 25 9899 9915 1368 CAGGTA 596819 749 765 GTTTCTCACTAeekkdddddddddkkee 38 9899 9915 1368 CAGGTA 596629 750 766 AGTTTCTCACTeekkddddddddkkeee 33 9900 9916 1369 ACAGGT 596820 750 766 AGTTTCTCACTeekkdddddddddkkee 50 9900 9916 1369 ACAGGT 596630 751 767 CAGTTTCTCACeekkddddddddkkeee 38 9901 9917 1370 TACAGG 596821 751 767 CAGTTTCTCACeekkdddddddddkkee 38 9901 9917 1370 TACAGG 596631 752 768 TCAGTTTCTCAeekkddddddddkkeee 25 9902 9918 1371 CTACAG 596822 752 768 TCAGTTTCTCAeekkdddddddddkkee 43 9902 9918 1371 CTACAG 596632 753 769 ATCAGTTTCTCeekkddddddddkkeee 31 9903 9919 1372 ACTACA 596823 753 769 ATCAGTTTCTCeekkdddddddddkkee 44 9903 9919 1372 ACTACA 596633 754 770 AATCAGTTTCTeekkddddddddkkeee 34 9904 9920 1373 CACTAC 596824 754 770 AATCAGTTTCTeekkdddddddddkkee 53 9904 9920 1373 CACTAC 596634 761 777 GATCATAAATeekkddddddddkkeee 34 9911 9927 1374 CAGTTTC 596825 761 777 GATCATAAATeekkdddddddddkkee 38 9911 9927 1374 CAGTTTC 596635 762 778 TGATCATAAAeekkddddddddkkeee 49 9912 9928 1375 TCAGTTT 596826 762 778 TGATCATAAAeekkdddddddddkkee 38 9912 9928 1375 TCAGTTT 596636 763 779 GTGATCATAAeekkddddddddkkeee 33 9913 9929 1376 ATCAGTT 596827 763 779 GTGATCATAAeekkdddddddddkkee 48 9913 9929 1376 ATCAGTT 596637 764 780 AGTGATCATAeekkddddddddkkeee 23 9914 9930 1377 AATCAGT 596828 764 780 AGTGATCATAeekkdddddddddkkee 32 9914 9930 1377 AATCAGT 596638 766 782 CAAGTGATCAeekkddddddddkkeee 47 9916 9932 1378 TAAATCA 596829 766 782 CAAGTGATCAeekkdddddddddkkee 29 9916 9932 1378 TAAATCA 596639 767 783 CCAAGTGATCeekkddddddddkkeee 40 9917 9933 1379 ATAAATC 596830 767 783 CCAAGTGATCeekkdddddddddkkee 48 9917 9933 1379 ATAAATC 596640 768 784 TCCAAGTGATeekkddddddddkkeee 42 9918 9934 1380 CATAAAT 596831 768 784 TCCAAGTGATeekkdddddddddkkee 39 9918 9934 1380 CATAAAT 596641 770 786 CTTCCAAGTGeekkddddddddkkeee 40 9920 9936 1381 ATCATAA 596832 770 786 CTTCCAAGTGeekkdddddddddkkee 54 9920 9936 1381 ATCATAA 596642 771 787 TCTTCCAAGTGeekkddddddddkkeee 33 9921 9937 1382 ATCATA 596833 771 787 TCTTCCAAGTGeekkdddddddddkkee 43 9921 9937 1382 ATCATA 596643 772 788 ATCTTCCAAGTeekkddddddddkkeee 38 9922 9938 1184 GATCAT 596834 772 788 ATCTTCCAAGTeekkdddddddddkkee 38 9922 9938 1184 GATCAT

TABLE 22 Percent inhibition of SOD-1 mRNA by deoxy, MOE and cEtargeting SEQ ID NO: 1 and/or 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1NO: 2 NO: 2 SEQ ISIS Start Stop % Start Stop ID NO Site Site SequenceChemistry inhibition Site Site NO 333611 167 186 CCGTCGCCCTeeeeeddddddddddeeeee 62 973 992 21 TCAGCACGCA 596720 167 183 TCGCCCTTCAeekkddddddddkkeee 53 973 989 966 GCACGCA 596911 167 183 TCGCCCTTCAeekkdddddddddkkee 58 973 989 966 GCACGCA 596644 773 789 AATCTTCCAAeekkddddddddkkeee 19 9923 9939 1185 GTGATCA 596835 773 789 AATCTTCCAAeekkdddddddddkkee 38 9923 9939 1185 GTGATCA 596645 774 790 AAATCTTCCAeekkddddddddkkeee 46 9924 9940 1186 AGTGATC 596836 774 790 AAATCTTCCAeekkdddddddddkkee 48 9924 9940 1186 AGTGATC 596646 782 798 AACTATACAAeekkddddddddkkeee 60 9932 9948 1193 ATCTTCC 596837 782 798 AACTATACAAeekkdddddddddkkee 63 9932 9948 1193 ATCTTCC 596647 783 799 AAACTATACAeekkddddddddkkeee 55 9933 9949 1194 AATCTTC 596838 783 799 AAACTATACAeekkdddddddddkkee 55 9933 9949 1194 AATCTTC 596648 806 822 AGACATTTTAeekkddddddddkkeee 46 9956 9972 1383 ACTGAGT 596839 806 822 AGACATTTTAeekkdddddddddkkee 53 9956 9972 1383 ACTGAGT 596649 817 833 GTCATTGAAAeekkddddddddkkeee 2 9967 9983 1201 CAGACAT 596840 817 833 GTCATTGAAAeekkdddddddddkkee 15 9967 9983 1201 CAGACAT 596650 819 835 AGGTCATTGAeekkddddddddkkeee 40 9969 9985 1203 AACAGAC 596841 819 835 AGGTCATTGAeekkdddddddddkkee 44 9969 9985 1203 AACAGAC 596651 822 838 TACAGGTCATeekkddddddddkkeee 26 9972 9988 1206 TGAAACA 596842 822 838 TACAGGTCATeekkdddddddddkkee 38 9972 9988 1206 TGAAACA 596652 823 839 ATACAGGTCAeekkddddddddkkeee 33 9973 9989 1207 TTGAAAC 596843 823 839 ATACAGGTCAeekkdddddddddkkee 22 9973 9989 1207 TTGAAAC 596653 825 841 AAATACAGGTeekkddddddddkkeee 28 9975 9991 1209 CATTGAA 596844 825 841 AAATACAGGTeekkdddddddddkkee 47 9975 9991 1209 CATTGAA 596654 827 843 CAAAATACAGeekkddddddddkkeee 44 9977 9993 1211 GTCATTG 596845 827 843 CAAAATACAGeekkdddddddddkkee 56 9977 9993 1211 GTCATTG 596655 830 846 TGGCAAAATAeekkddddddddkkeee 33 9980 9996 1214 CAGGTCA 596846 830 846 TGGCAAAATAeekkdddddddddkkee 43 9980 9996 1214 CAGGTCA 596656 831 847 CTGGCAAAATeekkddddddddkkeee 25 9981 9997 1215 ACAGGTC 596847 831 847 CTGGCAAAATeekkdddddddddkkee 53 9981 9997 1215 ACAGGTC 596657 833 849 GTCTGGCAAAeekkddddddddkkeee 30 9983 9999 1217 ATACAGG 596848 833 849 GTCTGGCAAAeekkdddddddddkkee 38 9983 9999 1217 ATACAGG 596658 836 852 TAAGTCTGGCeekkddddddddkkeee 24 9986 10002 1220 AAAATAC 596849 836 852 TAAGTCTGGCeekkdddddddddkkee 46 9986 10002 1220 AAAATAC 596659 837 853 TTAAGTCTGGeekkddddddddkkeee 27 9987 10003 1221 CAAAATA 596850 837 853 TTAAGTCTGGeekkdddddddddkkee 42 9987 10003 1221 CAAAATA 596660 840 856 GATTTAAGTCeekkddddddddkkeee 19 9990 10006 1384 TGGCAAA 596851 840 856 GATTTAAGTCeekkdddddddddkkee 35 9990 10006 1384 TGGCAAA 596661 841 857 TGATTTAAGTeekkddddddddkkeee 52 9991 10007 1385 CTGGCAA 596852 841 857 TGATTTAAGTeekkdddddddddkkee 52 9991 10007 1385 CTGGCAA 596662 842 858 GTGATTTAAGeekkddddddddkkeee 54 9992 10008 1386 TCTGGCA 596853 842 858 GTGATTTAAGeekkdddddddddkkee 69 9992 10008 1386 TCTGGCA 596663 843 859 TGTGATTTAAeekkddddddddkkeee 45 9993 10009 1387 GTCTGGC 596854 843 859 TGTGATTTAAeekkdddddddddkkee 58 9993 10009 1387 GTCTGGC 596664 844 860 CTGTGATTTAeekkddddddddkkeee n.d. 9994 10010 1388 AGTCTGG 596855 844 860 CTGTGATTTAeekkdddddddddkkee 61 9994 10010 1388 AGTCTGG 596665 845 861 TCTGTGATTTeekkddddddddkkeee 49 9995 10011 1389 AAGTCTG 596856 845 861 TCTGTGATTTeekkdddddddddkkee 49 9995 10011 1389 AAGTCTG 596666 846 862 ATCTGTGATTeekkddddddddkkeee 35 9996 10012 1390 TAAGTCT 596857 846 862 ATCTGTGATTeekkdddddddddkkee 37 9996 10012 1390 TAAGTCT 596667 847 863 CATCTGTGATeekkddddddddkkeee 42 9997 10013 1391 TTAAGTC 596858 847 863 CATCTGTGATeekkdddddddddkkee 48 9997 10013 1391 TTAAGTC 596668 848 864 CCATCTGTGAeekkddddddddkkeee 46 9998 10014 1392 TTTAAGT 596859 848 864 CCATCTGTGAeekkdddddddddkkee 47 9998 10014 1392 TTTAAGT 596669 849 865 CCCATCTGTGeekkddddddddkkeee 49 9999 10015 1393 ATTTAAG 596860 849 865 CCCATCTGTGeekkdddddddddkkee 49 9999 10015 1393 ATTTAAG 596670 850 866 ACCCATCTGTeekkddddddddkkeee 33 10000 10016 1394 GATTTAA 596861 850 866 ACCCATCTGTeekkdddddddddkkee 44 10000 10016 1394 GATTTAA 596671 851 867 TACCCATCTGeekkddddddddkkeee 29 10001 10017 1395 TGATTTA 596862 851 867 TACCCATCTGeekkdddddddddkkee 45 10001 10017 1395 TGATTTA 596672 854 870 TAATACCCATeekkddddddddkkeee 25 10004 10020 1223 CTGTGAT 596863 854 870 TAATACCCATeekkdddddddddkkee 28 10004 10020 1223 CTGTGAT 596673 855 871 TTAATACCCAeekkddddddddkkeee 28 10005 10021 1224 TCTGTGA 596864 855 871 TTAATACCCAeekkdddddddddkkee 26 10005 10021 1224 TCTGTGA 596674 858 874 AGTTTAATACeekkddddddddkkeee 29 10008 10024 1227 CCATCTG 596865 858 874 AGTTTAATACeekkdddddddddkkee 43 10008 10024 1227 CCATCTG 596675 859 875 AAGTTTAATAeekkddddddddkkeee 54 10009 10025 1228 CCCATCT 596866 859 875 AAGTTTAATAeekkdddddddddkkee 59 10009 10025 1228 CCCATCT 596676 860 876 CAAGTTTAATeekkddddddddkkeee 52 10010 10026 1229 ACCCATC 596867 860 876 CAAGTTTAATeekkdddddddddkkee 62 10010 10026 1229 ACCCATC 596677 861 877 ACAAGTTTAAeekkddddddddkkeee 58 10011 10027 1230 TACCCAT 596868 861 877 ACAAGTTTAAeekkdddddddddkkee 61 10011 10027 1230 TACCCAT 596678 862 878 GACAAGTTTAeekkddddddddkkeee 54 10012 10028 1231 ATACCCA 596869 862 878 GACAAGTTTAeekkdddddddddkkee 59 10012 10028 1231 ATACCCA 596679 863 879 TGACAAGTTTeekkddddddddkkeee 43 10013 10029 1232 AATACCC 596870 863 879 TGACAAGTTTeekkdddddddddkkee 52 10013 10029 1232 AATACCC 596680 864 880 CTGACAAGTTeekkddddddddkkeee 30 10014 10030 1233 TAATACC 596871 864 880 CTGACAAGTTeekkdddddddddkkee 36 10014 10030 1233 TAATACC 596681 865 881 TCTGACAAGTeekkddddddddkkeee 33 10015 10031 1234 TTAATAC 596872 865 881 TCTGACAAGTeekkdddddddddkkee 20 10015 10031 1234 TTAATAC

TABLE 23 Percent inhibition of SOD-1 mRNA by deoxy, MOE and cEtgapmers targeting SEQ ID NO: 1 and/or 2 SEQ SEQ SEQ SEQ ID ID ID IDNO: 1 NO: 1 NO: 2 NO: 2 SEQ ISIS Start Stop % Start Stop ID NO Site SiteSequence Chemistry inhibition Site Site NO 333611 167 186 CCGTCGCCCTeeeeeddddddddddeeeee 68 973 992 21 TCAGCACGCA 596720 167 183 TCGCCCTTCAeekkddddddddkkeee 64 973 989 966 GCACGCA 596911 167 183 TCGCCCTTCAeekkdddddddddkkee 71 973 989 966 GCACGCA 596682 884 900 GCTTGAATGAeekkddddddddkkeee 24 10034 10050 1396 CAAAGAA 596873 884 900 GCTTGAATGAeekkdddddddddkkee 32 10034 10050 1396 CAAAGAA 596683 885 901 GGCTTGAATGeekkddddddddkkeee 54 10035 10051 1397 ACAAAGA 596874 885 901 GGCTTGAATGeekkdddddddddkkee 44 10035 10051 1397 ACAAAGA 596684 889 905 CACAGGCTTGeekkddddddddkkeee 34 10039 10055 1398 AATGACA 596875 889 905 CACAGGCTTGeekkdddddddddkkee 47 10039 10055 1398 AATGACA 596685 890 906 TCACAGGCTTeekkddddddddkkeee 28 10040 10056 1399 GAATGAC 596876 890 906 TCACAGGCTTeekkdddddddddkkee 46 10040 10056 1399 GAATGAC 596686 891 907 TTCACAGGCTeekkddddddddkkeee 20 10041 10057 1242 TGAATGA 596877 891 907 TTCACAGGCTeekkdddddddddkkee 16 10041 10057 1242 TGAATGA 596687 892 908 ATTCACAGGCeekkddddddddkkeee 19 10042 10058 1243 TTGAATG 596878 892 908 ATTCACAGGCeekkdddddddddkkee 29 10042 10058 1243 TTGAATG 596688 893 909 TATTCACAGGeekkddddddddkkeee 24 10043 10059 1244 CTTGAAT 596879 893 909 TATTCACAGGeekkdddddddddkkee 11 10043 10059 1244 CTTGAAT 596689 894 910 TTATTCACAGeekkddddddddkkeee 26 10044 10060 1245 GCTTGAA 596880 894 910 TTATTCACAGeekkdddddddddkkee 30 10044 10060 1245 GCTTGAA 596690 895 911 TTTATTCACAeekkddddddddkkeee 44 10045 10061 1246 GGCTTGA 596881 895 911 TTTATTCACAeekkdddddddddkkee 55 10045 10061 1246 GGCTTGA 596691 896 912 TTTTATTCACeekkddddddddkkeee 43 10046 10062 1247 AGGCTTG 596882 896 912 TTTTATTCACeekkdddddddddkkee 48 10046 10062 1247 AGGCTTG 596692 899 915 GGTTTTTATTeekkddddddddkkeee 38 10049 10065 1250 CACAGGC 596883 899 915 GGTTTTTATTeekkdddddddddkkee 57 10049 10065 1250 CACAGGC 596693 903 919 ACAGGGTTTTeekkddddddddkkeee 29 10053 10069 1254 TATTCAC 596884 903 919 ACAGGGTTTTeekkdddddddddkkee 47 10053 10069 1254 TATTCAC 596694 904 920 TACAGGGTTTeekkddddddddkkeee 13 10054 10070 1255 TTATTCA 596885 904 920 TACAGGGTTTeekkdddddddddkkee 31 10054 10070 1255 TTATTCA 596695 907 923 CCATACAGGGeekkddddddddkkeee 13 10057 10073 1258 TTTTTAT 596886 907 923 CCATACAGGGeekkdddddddddkkee 34 10057 10073 1258 TTTTTAT 596696 908 924 GCCATACAGGeekkddddddddkkeee 13 10058 10074 1259 GTTTTTA 596887 908 924 GCCATACAGGeekkdddddddddkkee 26 10058 10074 1259 GTTTTTA 596697 909 925 TGCCATACAGeekkddddddddkkeee 21 10059 10075 1260 GGTTTTT 596888 909 925 TGCCATACAGeekkdddddddddkkee 22 10059 10075 1260 GGTTTTT 596698 910 926 GTGCCATACAeekkddddddddkkeee 20 10060 10076 1261 GGGTTTT 596889 910 926 GTGCCATACAeekkdddddddddkkee 28 10060 10076 1261 GGGTTTT 596699 911 927 AGTGCCATACeekkddddddddkkeee 20 10061 10077 1262 AGGGTTT 596890 911 927 AGTGCCATACeekkdddddddddkkee 27 10061 10077 1262 AGGGTTT 596700 912 928 AAGTGCCATAeekkddddddddkkeee 15 10062 10078 1400 CAGGGTT 596891 912 928 AAGTGCCATAeekkdddddddddkkee 21 10062 10078 1400 CAGGGTT 596701 913 929 TAAGTGCCATeekkddddddddkkeee 26 10063 10079 1401 ACAGGGT 596892 913 929 TAAGTGCCATeekkdddddddddkkee 35 10063 10079 1401 ACAGGGT 596702 914 930 ATAAGTGCCAeekkddddddddkkeee 36 10064 10080 1402 TACAGGG 596893 914 930 ATAAGTGCCAeekkdddddddddkkee 46 10064 10080 1402 TACAGGG 596703 915 931 AATAAGTGCCeekkddddddddkkeee 40 10065 10081 1403 ATACAGG 596894 915 931 AATAAGTGCCeekkdddddddddkkee 36 10065 10081 1403 ATACAGG 596704 916 932 TAATAAGTGCeekkddddddddkkeee 22 10066 10082 1404 CATACAG 596895 916 932 TAATAAGTGCeekkdddddddddkkee 30 10066 10082 1404 CATACAG 596705 917 933 ATAATAAGTGeekkddddddddkkeee 27 10067 10083 1405 CCATACA 596896 917 933 ATAATAAGTGeekkdddddddddkkee 27 10067 10083 1405 CCATACA 596706 918 934 CATAATAAGTeekkddddddddkkeee 32 10068 10084 1406 GCCATAC 596897 918 934 CATAATAAGTeekkdddddddddkkee 34 10068 10084 1406 GCCATAC 596707 919 935 TCATAATAAGeekkddddddddkkeee 28 10069 10085 1407 TGCCATA 596898 919 935 TCATAATAAGeekkdddddddddkkee 34 10069 10085 1407 TGCCATA 596708 920 936 CTCATAATAAeekkddddddddkkeee 30 10070 10086 1408 GTGCCAT 596899 920 936 CTCATAATAAeekkdddddddddkkee 44 10070 10086 1408 GTGCCAT 596709 921 937 CCTCATAATAeekkddddddddkkeee 29 10071 10087 1409 AGTGCCA 596900 921 937 CCTCATAATAeekkdddddddddkkee 31 10071 10087 1409 AGTGCCA 596710 922 938 GCCTCATAATeekkddddddddkkeee 41 10072 10088 1410 AAGTGCC 596901 922 938 GCCTCATAATeekkdddddddddkkee 33 10072 10088 1410 AAGTGCC 596711 923 939 AGCCTCATAAeekkddddddddkkeee 16 10073 10089 1411 TAAGTGC 596902 923 939 AGCCTCATAAeekkdddddddddkkee 11 10073 10089 1411 TAAGTGC 596712 924 940 TAGCCTCATAeekkddddddddkkeee 11 10074 10090 1412 ATAAGTG 596903 924 940 TAGCCTCATAeekkdddddddddkkee 27 10074 10090 1412 ATAAGTG 596713 925 941 ATAGCCTCATeekkddddddddkkeee 20 10075 10091 1413 AATAAGT 596904 925 941 ATAGCCTCATeekkdddddddddkkee 27 10075 10091 1413 AATAAGT 596714 926 942 AATAGCCTCAeekkddddddddkkeee 20 10076 10092 1414 TAATAAG 596905 926 942 AATAGCCTCAeekkdddddddddkkee 25 10076 10092 1414 TAATAAG 596715 931 947 CTTTTAATAGeekkddddddddkkeee 45 10081 10097 1415 CCTCATA 596906 931 947 CTTTTAATAGeekkdddddddddkkee 34 10081 10097 1415 CCTCATA 596716 932 948 TCTTTTAATAeekkddddddddkkeee 52 10082 10098 1416 GCCTCAT 596907 932 948 TCTTTTAATAeekkdddddddddkkee 56 10082 10098 1416 GCCTCAT 596717 936 952 GGATTCTTTTeekkddddddddkkeee 14 10086 10102 1417 AATAGCC 596908 936 952 GGATTCTTTTeekkdddddddddkkee 19 10086 10102 1417 AATAGCC 596718 938 954 TTGGATTCTTeekkddddddddkkeee 23 10088 10104 1263 TTAATAG 596909 938 954 TTGGATTCTTeekkdddddddddkkee 8 10088 10104 1263 TTAATAG 596719 949 965 TTAGTTTGAAeekkddddddddkkeee 31 10099 10115 1418 TTTGGAT 596910 949 965 TTAGTTTGAAeekkdddddddddkkee 16 10099 10115 1418 TTTGGAT

Example 6: Dose-Dependent Inhibition of Human SOD-1 with ModifiedOligonucleotides in HepG2 Cells

Gapmers from the studies described above exhibiting significant in vitroinhibition of SOD-1 mRNA were selected and tested at various doses inHepG2 cells. Benchmark compound ISIS 333611 and other compoundspreviously disclosed in WO 2005/040180, including ISIS 146144, 146145,150445, 150446, 150447, 150454, 150463, 150465, 333606, 333609, and333611 were also tested.

The modified oligonucleotides were tested in a series of experimentsthat had similar culture conditions. The results for each experiment arepresented in separate tables shown below. Cells were plated at a densityof 20,000 cells per well and transfected using electroporation with0.813 μM, 1.625 μM, 3.250 μM, 6.500 μM, and 13.000 μM concentrations ofmodified oligonucleotide, as specified in the Tables below. After atreatment period of approximately 16 hours, RNA was isolated from thecells and SOD-1 mRNA levels were measured by quantitative real-time PCR.Human primer probe set RTS3898 was used to measure mRNA levels. SOD-1mRNA levels were adjusted according to total RNA content, as measured byRIBOGREEN®. Results are presented as percent inhibition of SOD-1,relative to untreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotideis also presented. SOD-1 mRNA levels were significantly reduced in adose-dependent manner in modified oligonucleotide treated cells.

TABLE 24 Dose-dependent inhibition of SOD-1 mRNA 13.000 IC₅₀ ISIS No0.813 μM 1.625 μM 3.250 μM 6.500 μM μM (μM) 150445 7 21 44 56 82 4.4150446 15 32 47 71 87 3.2 150447 26 43 68 81 93 2.0 150463 16 38 51 6685 3.1 333611 18 39 57 66 79 3.0 393336 18 34 53 69 83 3.1 393343 24 3253 73 42 5.1 436863 18 42 58 72 89 2.6 590089 28 59 70 82 90 1.5 59009034 56 76 82 51 1.1 590091 30 44 68 84 88 1.9 590094 16 35 57 73 76 3.0590113 34 35 57 73 84 2.3

TABLE 25 Dose-dependent inhibition of SOD-1 mRNA 13.000 IC₅₀ ISIS No0.813 μM 1.625 μM 3.250 μM 6.500 μM μM (μM) 150465 42 59 77 82 87 1.0333611 17 26 40 64 82 3.8 378879 14 35 63 72 86 2.8 393371 28 42 57 7480 2.3 489520 28 44 64 72 84 2.2 590177 53 59 69 85 88 0.7 590178 40 5371 73 87 1.3 590180 18 42 51 64 73 3.3 590187 34 51 68 80 92 1.6 59018830 46 61 76 88 2.0 590189 37 49 68 78 88 1.6 590190 38 58 77 84 89 1.1590191 29 56 71 77 84 1.6 590192 37 59 72 80 87 1.2

TABLE 26 Dose-dependent inhibition of SOD-1 mRNA 13.000 IC₅₀ ISIS No0.813 μM 1.625 μM 3.250 μM 6.500 μM μM (μM) 146144 15 58 67 78 64 2.2146145 31 53 67 81 90 1.6 333606 11 39 62 75 91 2.7 333609 13 37 57 7185 2.9 333611 14 30 53 68 86 3.2 590250 8 19 47 64 84 3.9 590626 61 7284 84 87 0.2 592630 24 33 58 70 85 2.7 592631 19 48 59 74 88 2.3 59264520 31 53 74 89 2.8 592649 14 32 56 69 82 3.2

TABLE 27 Dose-dependent inhibition of SOD-1 mRNA 13.000 IC₅₀ ISIS No0.813 μM 1.625 μM 3.250 μM 6.500 μM μM (μM) 150454 13 24 49 69 83 3.5333611 28 28 53 68 82 3.0 489505 13 24 38 56 81 4.5 489516 25 16 39 6179 4.3 592652 11 31 52 74 46 5.1 592714 8 25 45 69 82 3.8 592715 18 3550 70 83 3.1 592762 44 66 74 81 89 0.8 592763 50 68 74 86 95 0.7 59276426 43 48 76 87 2.5 592766 36 53 66 77 89 1.5 592767 25 31 54 70 79 2.9592769 35 31 56 73 80 2.5 592771 34 43 58 70 80 2.2

Example 7: Dose-Dependent Inhibition of Human SOD-1 with ModifiedOligonucleotides in HepG2 Cells

Gapmers from the studies described above exhibiting significant in vitroinhibition of SOD-1 mRNA were selected and tested at various doses inHepG2 cells. Benchmark compound, ISIS 333611, and ISIS 333625, both ofwhich were previously disclosed in WO 2005/040180 were also tested.

The modified oligonucleotides were tested in a series of experimentsthat had similar culture conditions. The results for each experiment arepresented in separate tables shown below. Cells were plated at a densityof 20,000 cells per well and transfected using electroporation with0.148 μM, 0.444 μM, 1.330 μM, 4.000 μM, and 12.000 μM concentrations ofmodified oligonucleotide, as specified in the Tables below. After atreatment period of approximately 16 hours, RNA was isolated from thecells and SOD-1 mRNA levels were measured by quantitative real-time PCR.Human primer probe sets RTS3898 or HTS90 was used to measure mRNAlevels. SOD-1 mRNA levels were adjusted according to total RNA content,as measured by RIBOGREEN®. Results are presented as percent inhibitionof SOD-1, relative to untreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotideis also presented. SOD-1 mRNA levels were significantly reduced in adose-dependent manner in modified oligonucleotide treated cells.

TABLE 28 Dose response assay with primer probe set RTS3898 12.000 IC₅₀ISIS No 0.148 μM 0.444 μM 1.330 μM 4.000 μM μM (μM) 333611 6 14 29 51 783.2 596911 8 12 23 54 74 3.6 596720 7 14 31 55 71 3.4 596800 44 60 75 8482 0.2 596801 33 49 69 79 83 0.5 596610 16 44 65 78 84 0.8 596799 20 4564 75 84 0.8 596609 17 54 65 75 81 0.7 596883 13 22 36 45 51 8.6 48952316 40 62 78 90 0.9 590181 5 17 46 70 89 1.7 436868 10 35 47 69 82 1.4596768 16 37 62 82 89 0.9 596775 36 50 66 83 89 0.4

TABLE 29 Dose response assay with primer probe set HTS90 12.000 IC₅₀ISIS No 0.148 μM 0.444 μM 1.330 μM 4.000 μM μM (μM) 333625 0 4 17 56 843.3 489532 54 70 78 86 96 0.1 590154 0 14 25 56 86 2.7 596173 0 5 25 6392 2.4 596174 7 12 37 46 84 2.8 596178 2 16 40 68 82 2.1 596179 0 17 4164 80 2.3 596308 0 5 22 56 80 3.3 596572 18 35 62 83 90 0.9 596589 10 4561 77 91 0.9 596600 41 56 71 85 92 0.3 596602 14 51 74 86 95 0.6 59678922 55 69 86 91 0.5 596795 16 43 64 82 93 0.8

Example 8: Dose-Dependent Inhibition of Human SOD-1 with ModifiedOligonucleotides in HepG2 Cells

Gapmers from the studies described above exhibiting significant in vitroinhibition of SOD-1 mRNA were selected and tested at various doses inHepG2 cells. Benchmark compound, ISIS 333611, and additional compoundsincluding, ISIS 146143, 150442, 195753, 333607, and 333608, werepreviously disclosed in WO 2005/040180 were also tested.

The modified oligonucleotides were tested in a series of experimentsthat had similar culture conditions. The results for each experiment arepresented in separate tables shown below. Cells were plated at a densityof 20,000 cells per well and transfected using electroporation with0.1875 μM, 0.7500 μM, 3.0000 μM, and 12.0000 μM concentrations ofmodified oligonucleotide, as specified in the Tables below. After atreatment period of approximately 16 hours, RNA was isolated from thecells and SOD-1 mRNA levels were measured by quantitative real-time PCR.Human primer probe sets RTS3898 was used to measure mRNA levels. SOD-1mRNA levels were adjusted according to total RNA content, as measured byRIBOGREEN®. Results are presented as percent inhibition of SOD-1,relative to untreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotideis also presented. SOD-1 mRNA levels were significantly reduced in adose-dependent manner in modified oligonucleotide treated cells.

TABLE 30 Dose-dependent inhibition of SOD-1 mRNA ISIS 0.1875 0.75 3.0012.00 IC₅₀ No μM μM μM μM (μM) 333611 0 27 60 91 2 596572 38 65 87 960.3 596583 62 89 95 95 <0.1 596590 40 79 89 94 0.2 596602 40 75 92 980.2 596603 51 79 92 96 0.1 596604 48 78 91 95 0.1 596605 50 86 90 97 0.1596764 11 67 89 94 0.7 596768 27 49 82 92 0.7 596773 32 62 89 98 0.4596774 56 89 93 96 <0.1 596775 31 75 90 97 0.3 596780 24 71 85 98 0.5596781 30 80 93 97 0.3 596791 38 74 89 95 0.3 596794 43 75 91 97 0.2596795 28 66 91 98 0.4 596796 43 78 93 98 0.2

TABLE 31 Dose-dependent inhibition of SOD-1 mRNA ISIS 0.1875 0.75 3.0012.00 IC₅₀ No μM μM μM μM (μM) 333611 0 15 68 95 2.1 596589 35 70 90 970.3 596789 38 71 89 97 0.3 596600 41 73 89 95 0.2 596582 30 71 87 95 0.4596784 26 68 89 95 0.4 596787 44 67 89 94 0.2 596779 29 71 89 97 0.4596792 37 63 83 95 0.4 596782 27 61 85 96 0.5 596765 34 59 87 95 0.4596793 27 65 88 96 0.5 596570 25 60 84 91 0.6 596769 21 64 85 96 0.6596783 10 57 84 94 0.9 596584 26 67 84 93 0.5 596571 37 71 81 92 0.3596598 30 62 81 94 0.5 596588 11 64 87 95 0.7

TABLE 32 Dose-dependent inhibition of SOD-1 mRNA ISIS 0.1875 0.75 3.0012.00 IC₅₀ No μM μM μM μM (μM) 146143 6 12 51 88 2.5 150442 10 21 39 902.5 195753 13 23 48 77 2.8 333607 17 26 59 83 1.9 333608 0 2 28 92 3.7333611 0 13 52 91 2.4 596573 26 51 77 91 0.8 596577 32 55 78 93 0.6596591 23 51 74 91 0.8 596592 18 48 66 86 1.1 596593 16 58 78 87 0.8596596 4 49 72 87 1.3 596761 28 55 74 91 0.7 596762 0 47 75 90 1.3596763 0 40 78 92 1.5 596766 4 50 68 86 1.3 596785 10 47 77 91 1.1596786 0 45 75 97 1.3 596788 9 52 81 95 1

TABLE 33 Dose-dependent inhibition of SOD-1 mRNA ISIS 0.1875 0.75 3.0012.00 IC₅₀ No μM μM μM μM (μM) 333611 3 22 60 92 2 596302 9 27 59 89 1.9596308 29 47 82 96 0.7 596309 13 42 75 92 1.1 596310 13 16 48 81 2.8596313 15 37 70 88 1.3 596314 18 45 74 92 1 596606 55 78 87 93 0.1596607 44 71 83 84 0.2 596608 46 74 84 81 0.1 596609 30 61 79 87 0.5596610 39 69 82 86 0.3 596612 16 50 62 77 1.4 596797 67 84 94 96 <0.1596798 42 68 86 89 0.2 596799 35 66 83 87 0.4 596800 45 73 84 87 0.2596801 40 67 86 88 0.3 596803 28 41 63 71 1.4

TABLE 34 Dose-dependent inhibition of SOD-1 mRNA ISIS 0.1875 0.75 3.0012.00 IC₅₀ No μM μM μM μM (μM) 333611 0 30 56 69 3.1 590475 19 39 69 881.2 590625 19 51 74 85 1.0 590626 42 72 88 90 0.2 590627 16 42 70 84 1.0590634 45 72 86 92 0.2 590635 39 60 80 90 0.4 590644 44 62 80 86 0.3590650 34 56 82 93 0.5 590653 52 78 86 85 0.1 590655 35 71 79 82 0.3596530 25 22 72 79 2.0 596531 8 38 74 96 1.2 596559 15 36 79 95 1.1596721 14 47 82 97 0.9 596723 12 47 79 94 1.0 596726 24 42 80 94 0.9596735 7 32 77 96 1.3 596736 25 52 82 97 0.7

Example 9: Dose-Dependent Inhibition of Human SOD-1 in HepG2 Cells byGapmers with Mixed Backbone Chemistry

Additional gapmers were designed based on the sequences of theoligonucleotides disclosed in studies described above. Theoligonucleotides were designed as 5-10-5 MOE, 5-8-5 MOE, and deoxy, MOEand cEt oligonucleotides. The 5-10-5 MOE gapmers are 20 nucleosides inlength, wherein the central gap segment is comprised of ten2′-deoxyribonucleosides and is flanked by wing segments on the 5′direction and the 3′ direction comprising five nucleosides each. The5-8-5 MOE gapmers are 18 nucleosides in length, wherein the central gapsegment is comprised of eight 2′-deoxyribonucleosides and is flanked bywing segments on the 5′ direction and the 3′ direction comprising fivenucleosides each. Each nucleoside in the 5′ wing segment and eachnucleoside in the 3′ wing segment has a 2′-MOE modification. The deoxy,MOE and cEt oligonucleotides are 16 or 17 nucleosides in length whereineach nucleoside has a MOE sugar modification, a cEt sugar modification,or a deoxy moiety. The sugar chemistry of each oligonucleotide isdenoted as in the Chemistry column, where ‘k’ indicates a cEt modifiedsugar; ‘d’ indicates a 2′-deoxyribose; and ‘e’ indicates a 2′-MOEmodified sugar. The internucleoside linkages throughout each gapmer areeither phosphodiester or phosphorothioate linkages. The internucleosidelinkages of each oligonucleotide are denoted in the Backbone Chemistrycolumn, where ‘o’ indicates a phosphodiester linkage and ‘s’ denotes aphosphorothioate linkage. All cytosine residues throughout each gapmerare 5-methylcytosines. “Start site” indicates the 5′-most nucleoside towhich the gapmer is targeted in the human gene sequence. “Stop site”indicates the 3′-most nucleoside to which the gapmer is targeted humangene sequence. Each gapmer listed in the Tables below is targeted toeither the human SOD-1 mRNA, designated herein as SEQ ID NO: 1 (GENBANKAccession No. NM_000454.4) or the human SOD-1 genomic sequence,designated herein as SEQ ID NO: 2 (GENBANK Accession No. NT_011512.10truncated from nucleotides 18693000 to Ser. No. 18/704,000).

TABLE 35Modified oligonucleotides targeting human SOD-1 with mixed backbone chemistrySEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 SEQ ISIS Start StopBackbone Start Stop ID NO Site Site Sequence Sugar ModificationsChemistry Site Site NO 611458 226 245 ACACCTTCAC eeeeeddddddddddeeeeesooosssssssssssooos 4980 4999 23 TGGTCCATTA 654335 233 248 CCCACACCTTekddddddddekekee sossssssssoooss 4987 5002 1420 CACTGG 611474 234 253GCTTCCCCAC eeeeeddddddddddeeeee sooosssssssssssooos 4988 5007 149ACCTTCACTG 654301 234 251 TTCCCCACAC eeeeeddddddddeeeeesooosssssssssooss 4988 5005 1421 CTTCACTG 654336 234 249 CCCCACACCTekddddddddekekee sossssssssoooss 4988 5003 1422 TCACTG 654302 235 252CTTCCCCACA eeeeeddddddddeeeee sooosssssssssooss 4989 5006 1423 CCTTCACT654319 235 250 TCCCCACACC kekeddddddddekek sooossssssssoss 4989 50041424 TTCACT 654337 235 250 TCCCCACACC ekddddddddekekee sossssssssoooss4989 5004 1424 TTCACT 654303 236 253 GCTTCCCCAC eeeeeddddddddeeeeesooosssssssssooss 4990 5007 1425 ACCTTCAC 654320 236 251 TTCCCCACACkekeddddddddekek sooossssssssoss 4990 5005 1426 CTTCAC 654321 237 252CTTCCCCACA kekeddddddddekek sooossssssssoss 4991 5006 1427 CCTTCA 611475321 340 GTGAGGACCT eeeeeddddddddddeeeee sooosssssssssssooos 7637 7656172 GCACTGGTAC 611460 588 607 GGCGATCCCA eeeeeddddddddddeeeeesooosssssssssssooos 9738 9757 47 ATTACACCAC 654304 663 680 ATACATTTCTeeeeeddddddddeeeee sooosssssssssooss 9813 9830 1429 ACAGCTAG 654305 664681 GATACATTTC eeeeeddddddddeeeee sooosssssssssooss 9814 9831 1430TACAGCTA 654340 664 679 TACATTTCTA ekddddddddekekee sossssssssoooss 98149829 1431 CAGCTA 611492 665 684 CAGGATACAT eeeeeddddddddddeeeeesooosssssssssssooos 9815 9834 725 TTCTACAGCT 654306 665 682 GGATACATTTeeeeeddddddddeeeee sooosssssssssooss 9815 9832 1432 CTACAGCT 654323 665680 ATACATTTCT kekeddddddddekek sooossssssssoss 9815 9830 1433 ACAGCT654341 665 680 ATACATTTCT ekddddddddekekee sossssssssoooss 9815 98301433 ACAGCT 611500 666 685 TCAGGATACA eeeeeddddddddddeeeeesooosssssssssssooos 9816 9835 823 TTTCTACAGC 612941 666 682 GGATACATTTeekkdddddddddkkee sooosssssssssoss 9816 9832 1342 CTACAGC 654307 666 683AGGATACATT eeeeeddddddddeeeee sooosssssssssooss 9816 9833 1434 TCTACAGC654324 666 681 GATACATTTC kekeddddddddekek sooossssssssoss 9816 98311435 TACAGC 654342 666 681 GATACATTTC ekddddddddekekee sossssssssoooss9816 9831 1435 TACAGC 654308 667 684 CAGGATACAT eeeeeddddddddeeeeesooosssssssssooss 9817 9834 1436 TTCTACAG 612925 676 692 ATGTTTATCAeekkddddddddkkeee soosssssssssooss 9826 9842 1348 GGATACA 612944 676 692ATGTTTATCA eekkdddddddddkkee sooosssssssssoss 9826 9842 1348 GGATACA654343 677 692 ATGTTTATCA ekddddddddekekee sossssssssoooss 9827 98421437 GGATAC 612927 678 694 TAATGTTTAT eekkddddddddkkeee soosssssssssooss9828 9844 1350 CAGGATA 654309 678 695 TTAATGTTTA eeeeeddddddddeeeeesooosssssssssooss 9828 9845 1438 TCAGGATA 612928 679 695 TTAATGTTTAeekkddddddddkkeee soosssssssssooss 9829 9845 1351 TCAGGAT 654310 679 696TTTAATGTTT eeeeeddddddddeeeee sooosssssssssooss 9829 9846 1439 ATCAGGAT654311 680 697 GTTTAATGTT eeeeeddddddddeeeee sooosssssssssooss 9830 98471440 TATCAGGA 654327 680 695 TTAATGTTTA kekeddddddddekek sooossssssssoss9830 9845 1441 TCAGGA 654346 680 695 TTAATGTTTA ekddddddddekekeesossssssssoooss 9830 9845 1441 TCAGGA 611497 681 700 AGTGTTTAATeeeeeddddddddddeeeee sooosssssssssssooos 9831 9850 733 GTTTATCAGG 612948681 697 GTTTAATGTT eekkdddddddddkkee sooosssssssssoss 9831 9847 1352TATCAGG 654312 681 698 TGTTTAATGT eeeeeddddddddeeeee sooosssssssssooss9831 9848 1442 TTATCAGG 654328 681 696 TTTAATGTTT kekeddddddddekeksooossssssssoss 9831 9846 1443 ATCAGG 654347 681 696 TTTAATGTTTekddddddddekekee sossssssssoooss 9831 9846 1443 ATCAGG 654313 682 699GTGTTTAATG eeeeeddddddddeeeee sooosssssssssooss 9832 9849 1444 TTTATCAG654329 682 697 GTTTAATGTT kekeddddddddekek sooossssssssoss 9832 98471445 TATCAG 654348 682 697 GTTTAATGTT ekddddddddekekee sossssssssoooss9832 9847 1445 TATCAG 612949 683 699 GTGTTTAATG eekkdddddddddkkeesooosssssssssoss 9833 9849 1172 TTTATCA 654314 683 700 AGTGTTTAATeeeeeddddddddeeeee sooosssssssssooss 9833 9850 1446 GTTTATCA 654330 683698 TGTTTAATGT kekeddddddddekek sooossssssssoss 9833 9848 1447 TTATCA612931 684 700 AGTGTTTAAT eekkddddddddkkeee soosssssssssooss 9834 98501173 GTTTATC 654315 684 701 CAGTGTTTAA eeeeeddddddddeeeeesooosssssssssooss 9834 9851 1448 TGTTTATC 654331 684 699 GTGTTTAATGkekeddddddddekek sooossssssssoss 9834 9849 1449 TTTATC 654350 684 699GTGTTTAATG ekddddddddekekee sossssssssoooss 9834 9849 1449 TTTATC 654316685 702 ACAGTGTTTA eeeeeddddddddeeeee sooosssssssssooss 9835 9852 1450ATGTTTAT 612918 686 702 ACAGTGTTTA eeekkdddddddkkeee soosssssssssooss9836 9852 1175 ATGTTTA 612932 686 702 ACAGTGTTTA eekkddddddddkkeeesoosssssssssooss 9836 9852 1175 ATGTTTA 654317 686 703 TACAGTGTTTeeeeeddddddddeeeee sooosssssssssooss 9836 9853 1451 AATGTTTA 654333 686701 CAGTGTTTAA kekeddddddddekek sooossssssssoss 9836 9851 1452 TGTTTA654352 686 701 CAGTGTTTAA ekddddddddekekee sossssssssoooss 9836 98511452 TGTTTA 654318 687 704 TTACAGTGTT eeeeeddddddddeeeeesooosssssssssooss 9837 9854 1453 TAATGTTT 654334 687 702 ACAGTGTTTAkekeddddddddekek sooossssssssoss 9837 9852 1454 ATGTTT

The newly designed oligonucleotides were tested at various doses inHepG2 cells. The modified oligonucleotides were tested in a series ofexperiments that had similar culture conditions. The results for eachexperiment are presented in separate tables shown below. Cells wereplated at a density of 20,000 cells per well and transfected usingelectroporation with 0.222 μM, 0.667 μM, 2.000 μM, and 6.000 μMconcentrations of modified oligonucleotide, as specified in the Tablesbelow. After a treatment period of approximately 16 hours, RNA wasisolated from the cells and SOD-1 mRNA levels were measured byquantitative real-time PCR. Human primer probe sets RTS3898 was used tomeasure mRNA levels. SOD-1 mRNA levels were adjusted according to totalRNA content, as measured by RIBOGREEN®. Results are presented as percentinhibition of SOD-1, relative to untreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotideis also presented. SOD-1 mRNA levels were significantly reduced in adose-dependent manner in modified oligonucleotide treated cells.

TABLE 36 Dose response assay ISIS 0.222 0.667 2.000 6.000 IC₅₀ No μM μMμM μM μM 333611 0 28 53 77 1.9 611458 0 21 50 79 2.0 611460 24 34 55 791.3 611474 14 32 55 79 1.5 611475 0 16 35 70 3.0 611492 38 70 88 95 0.3611497 28 55 80 89 0.6 611500 25 50 73 92 0.7 612918 51 70 74 80 <0.2612925 53 73 90 89 <0.2 612927 64 89 92 94 <0.2 612928 67 90 94 97 <0.2612931 68 76 84 86 <0.2 612932 61 73 88 91 <0.2 612941 62 78 91 95 <0.2612944 47 71 82 92 0.2 612948 76 90 93 94 <0.2 612949 58 68 83 96 <0.2654301 7 4 17 42 >6.0

TABLE 37 Dose response assay ISIS 0.222 0.667 2.000 6.000 IC₅₀ No μM μMμM μM μM 333611 14 20 35 69 3.0 611458 11 27 36 68 2.9 654302 0 8 38 486.2 654303 8 29 46 76 1.9 654304 7 28 54 79 1.7 654305 28 59 73 85 0.6654306 38 62 82 94 0.4 654307 9 43 65 86 1.1 654308 14 31 54 84 1.4654309 0 17 47 72 2.4 654310 10 24 28 53 6.6 654311 45 73 78 87 0.2654312 14 39 59 77 1.3 654313 20 43 56 81 1.2 654314 33 58 74 86 0.5654315 21 47 64 84 0.9 654316 19 30 46 70 2.0 654317 13 46 57 70 1.4654318 17 42 54 76 1.4

TABLE 38 Dose response assay ISIS 0.222 0.667 2.000 6.000 IC₅₀ No μM μMμM μM μM 333611 14 19 50 73 2.1 611458 9 22 39 72 2.5 654319 19 9 31 615.1 654320 6 16 20 59 5.9 654321 8 14 51 76 2.1 654323 55 73 89 95 <0.2654324 54 78 89 96 <0.2 654327 53 82 91 96 <0.2 654328 73 90 93 97 <0.2654329 58 78 86 94 <0.2 654330 42 54 69 86 0.4 654331 53 78 82 90 <0.2654333 50 67 81 86 0.2 654334 55 68 78 88 <0.2 654335 15 31 58 81 1.4654336 21 36 60 75 1.3 654337 16 34 58 80 1.4 654340 36 69 83 95 0.4654341 43 58 79 91 0.3

TABLE 39 Dose response assay ISIS 0.222 0.667 2.00 6.00 IC₅₀ No μM μM μMμM μM 333611 0 6 38 64 3.6 611458 3 14 36 63 3.6 654342 40 60 80 93 0.4654343 64 81 90 94 <0.2 654346 52 73 84 93 <0.2 654347 21 38 58 81 1.2654348 44 63 82 94 0.3 654350 40 63 76 86 0.3 654352 54 79 84 88 <0.2

Example 10: Dose-Dependent Inhibition of Human SOD-1 by Gapmers withMixed Backbone Chemistry

Additional gapmers were designed based on the sequences of theoligonucleotides disclosed in studies described above. Theoligonucleotides were designed as deoxy, MOE and cEt oligonucleotides.The deoxy, MOE and cEt oligonucleotides are 16 or 17 nucleosides inlength wherein each nucleoside has a MOE sugar modification, a cEt sugarmodification, or a deoxy moiety. The sugar chemistry of eacholigonucleotide is denoted as in the Chemistry column, where ‘k’indicates a cEt modified sugar; ‘d’ indicates a 2′-deoxyribose; and ‘e’indicates a 2′-MOE modified sugar. The internucleoside linkagesthroughout each gapmer are either phosphodiester or phosphorothioatelinkages. The internucleoside linkages of each oligonucleotide aredenoted in the Backbone Chemistry column, where ‘o’ indicates aphosphodiester linkage and ‘s’ indicates a phosphorothioate linkage. Allcytosine residues throughout each gapmer are 5-methylcytosines. “Startsite” indicates the 5′-most nucleoside to which the gapmer is targetedin the human gene sequence. “Stop site” indicates the 3′-most nucleosideto which the gapmer is targeted human gene sequence. Each gapmer listedin the Tables below is targeted to either the human SOD-1 mRNA,designated herein as SEQ ID NO: 1 (GENBANK Accession No. NM_000454.4) orthe human SOD-1 genomic sequence, designated herein as SEQ ID NO: 2(GENBANK Accession No. NT_011512.10 truncated from nucleotides 18693000to Ser. No. 18/704,000).

TABLE 40Modified oligonucleotides targeting human SOD-1 with mixed backbone chemistrySEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 SEQ ISIS Start StopBackbone Start Stop ID NO Site Site Sequence Sugar ModificationsChemistry Site Site NO 612916 664 680 ATACATTTCTA eeekkdddddddkkeeesoosssssssssooss 9814 9830 1170 CAGCTA 612947 679 695 TTAATGTTTATeekkdddddddddkkee sooosssssssssoss 9829 9845 1351 CAGGAT 654322 664 679TACATTTCTAC kekeddddddddekek sooossssssssoss 9814 9829 1431 AGCTA 654325667 682 GGATACATTT kekeddddddddekek sooossssssssoss 9817 9832 1455CTACAG 654326 679 694 TAATGTTTATC kekeddddddddekek sooossssssssoss 98299844 1456 AGGAT 654332 685 700 AGTGTTTAAT kekeddddddddekeksooossssssssoss 9835 9850 1457 GTTTAT 654338 662 677 CATTTCTACAGekddddddddekekee sossssssssoooss 9812 9827 1458 CTAGC 654339 663 678ACATTTCTACA ekddddddddekekee sossssssssoooss 9813 9828 1459 GCTAG 654344678 693 AATGTTTATCA ekddddddddekekee sossssssssoooss 9828 9843 1460GGATA 654345 679 694 TAATGTTTATC ekddddddddekekee sossssssssoooss 98299844 1456 AGGAT 654349 683 698 TGTTTAATGTT ekddddddddekekeesossssssssoooss 9833 9848 1447 TATCA 654351 685 700 AGTGTTTAATekddddddddekekee sossssssssoooss 9835 9850 1457 GTTTAT

The newly designed oligonucleotides were tested at various doses in A431cells. The modified oligonucleotides were tested in a series ofexperiments that had similar culture conditions. The results for eachexperiment are presented in separate tables shown below. Cells wereplated at a density of 5,000 cells per well and modifiedoligonucleotides were added to the media at 0.12 μM, 0.60 μM, 3.00 μM,and 15.00 μM concentrations of modified oligonucleotide for free uptakeby the cells, as specified in the Tables below. After a treatment periodof approximately 16 hours, RNA was isolated from the cells and SOD-1mRNA levels were measured by quantitative real-time PCR. Human primerprobe sets RTS3898 was used to measure mRNA levels. SOD-1 mRNA levelswere adjusted according to total RNA content, as measured by RIBOGREEN®.Results are presented as percent inhibition of SOD-1, relative tountreated control cells.

TABLE 41 Dose response assay ISIS 0.12 0.60 3.00 15.00 No μM μM μM μM333611 0 7 17 31 611458 5 4 4 10 612916 4 13 26 37 612918 12 26 45 47612944 1 4 21 29 612947 12 48 54 72 654305 7 15 39 52 654306 0 25 29 50654313 1 15 36 50 654314 2 35 52 67 654321 0 8 8 18 654322 0 13 36 59654329 7 7 41 66 654330 6 14 15 32 654337 0 0 7 21 654338 3 3 2 11654345 1 9 22 46 654346 0 7 21 46

TABLE 42 Dose response assay ISIS 0.12 0.60 3.00 15.00 No μM μM μM μM333611 0 0 0 2 611460 0 0 0 30 611474 0 0 11 0 612925 2 4 12 52 612927 038 54 68 612948 25 69 89 95 612949 22 57 73 84 654307 42 23 26 45 6543082 31 9 18 654315 0 8 39 52 654316 0 18 26 45 654323 15 14 16 52 65432412 22 21 34 654331 7 35 66 78 654332 2 31 47 61 654339 1 27 32 47 65434037 0 22 12 654347 20 5 12 33 654348 2 19 33 62

TABLE 43 Dose response assay ISIS 0.12 0.60 3.00 15.00 No μM μM μM μM333611 0 0 0 1 611475 0 17 0 16 611492 13 24 41 62 612928 12 36 49 72612931 31 68 83 86 654301 2 0 0 9 654302 0 8 3 0 654309 18 7 11 9 65431013 19 22 7 654317 3 0 1 20 654318 4 0 33 17 654325 0 0 0 14 654326 0 1517 48 654333 19 18 36 55 654334 0 0 0 6 654341 0 9 0 25 654342 0 0 0 18654349 0 13 31 49 654350 10 32 66 79

TABLE 44 Dose response assay ISIS 0.12 0.60 3.00 15.00 No μM μM μM μM333611 5 0 7 3 611497 16 49 60 75 611500 9 8 21 49 612932 17 8 26 37612941 4 12 36 51 654303 0 1 0 5 654304 9 10 27 43 654311 15 51 68 84654312 6 26 29 33 654319 3 44 2 8 654320 4 12 5 12 654327 3 45 65 81654328 15 44 73 85 654335 2 0 0 12 654336 0 0 0 0 654343 0 7 26 59654344 10 30 51 72 654351 10 22 48 77 654352 8 26 57 76

Example 11: Dose-Dependent Inhibition of Human SOD-1 by Gapmers withMixed Backbone Chemistry

Additional gapmers were designed based on the sequences of theoligonucleotides disclosed in studies described above. Theoligonucleotides were designed as 5-10-5 MOE gapmers, 4-8-5 MOE gapmers,5-8-5 MOE gapmers, 5-8-7 MOE gapmers, 6-8-6 MOE gapmers, 6-9-5 MOEgapmers, or deoxy, MOE and cEt oligonucleotides.

The 5-10-5 MOE gapmers are 20 nucleosides in length, wherein the centralgap segment is comprised of ten 2′-deoxynucleosides and is flanked bywing segments on the 5′ direction and the 3′ directions comprising fivenucleosides each. The 4-8-5 MOE gapmers are 17 nucleosides in length,wherein the central gap segment is comprised of eight2′-deoxyribonucleosides and is flanked by wing segments on the 5′direction and the 3′ directions comprising four and five nucleosidesrespectively. The 5-8-5 MOE gapmers are 18 nucleosides in length,wherein the central gap segment is comprised of eight2′-deoxynucleosides and is flanked by wing segments on the 5′ directionand the 3′ directions comprising five nucleosides each. The 5-8-7 MOEgapmers are 20 nucleosides in length, wherein the central gap segment iscomprised of eight 2′-deoxynucleosides and is flanked by wing segmentson the 5′ direction and the 3′ directions comprising five and sevennucleosides respectively. The 6-8-6 MOE gapmers are 20 nucleosides inlength, wherein the central gap segment is comprised of eight2′-deoxynucleosides and is flanked by wing segments on the 5′ directionand the 3′ directions comprising six nucleosides each. The 6-9-5 MOEgapmers are 20 nucleosides in length, wherein the central gap segment iscomprised of nine 2′-deoxynucleosides and is flanked by wing segments onthe 5′ direction and the 3′ directions comprising six and fivenucleosides respectively. Each nucleoside in the 5′ wing segment andeach nucleoside in the 3′ wing segment has a 2′-MOE modification.

The deoxy, MOE and cEt oligonucleotides are 17 nucleosides in lengthwherein each nucleoside has a MOE sugar modification, a cEt sugarmodification, or a deoxy moiety. The sugar chemistry of eacholigonucleotide is denoted as in the Chemistry column, where ‘k’indicates a cEt modified sugar; ‘d’ indicates a 2′-deoxyribose; and ‘e’indicates a 2′-MOE modified sugar.

The internucleoside linkages throughout each gapmer are eitherphosphodiester or phosphorothioate linkages. The internucleosidelinkages of each oligonucleotide are denoted in the Backbone Chemistrycolumn, where ‘o’ indicates a phosphodiester linkage and ‘s’ indicates aphosphorothioate linkage. All cytosine residues throughout each gapmerare 5-methylcytosines. “Start site” indicates the 5′-most nucleoside towhich the gapmer is targeted in the human gene sequence. “Stop site”indicates the 3′-most nucleoside to which the gapmer is targeted humangene sequence. Each gapmer listed in the Tables below is targeted toeither the human SOD-1 mRNA, designated herein as SEQ ID NO: 1 (GENBANKAccession No. NM_000454.4) or the human SOD-1 genomic sequence,designated herein as SEQ ID NO: 2 (GENBANK Accession No. NT 011512.10truncated from nucleotides 18693000 to Ser. No. 18/704,000).

TABLE 45Modified oligonucleotides targeting human SOD-1 with mixed backbone chemistrySEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 1 NO: 2 NO: 2 Start Stop BackboneStart Stop SEQ ID ISIS NO Site Site Sequence Motif Sugar ModificationsChemistry Site Site NO 666846 665 684 CAGGATACAT 5-10-5eeeeeddddddddddeeeee soooossssssssssooss 9815 9834 725 TTCTACAGCT MOE666849 665 684 CAGGATACAT 5-10-5 eeeeeddddddddddeeeeesooosssssssssssooss 9815 9834 725 TTCTACAGCT MOE 666853 665 684CAGGATACAT 5-10-5 eeeeeddddddddddeeeee sososssssssssssosos 9815 9834 725TTCTACAGCT MOE 666859 679 695 TTAATGTTTA Deoxy, eeeeddddddddkkeeesoosssssssssooss 9829 9845 1351 TCAGGAT MOE and cEt 666861 679 695TTAATGTTTA Deoxy, ekekddddddddeeeee soosssssssssooss 9829 9845 1351TCAGGAT MOE and cEt 666867 684 700 AGTGTTTAAT Deoxy, eekkddddddddeeeeesoosssssssssooss 9834 9850 1173 GTTTATC MOE and cEt 666869 684 700AGTGTTTAAT Deoxy, ekekddddddddkekee soosssssssssooss 9834 9850 1173GTTTATC MOE and cEt 666870 684 700 AGTGTTTAAT Deoxy, ekekddddddddeeeeesoosssssssssooss 9834 9850 1173 GTTTATC MOE and cEt 666919 666 682GGATACATTT Deoxy, eeeedddddddddkkee sooosssssssssoss 9816 9832 1342CTACAGC MOE and cEt 666921 666 682 GGATACATTT Deoxy, eeeeeddddddddkkeesooosssssssssoss 9816 9832 1342 CTACAGC MOE and cEt 666922 666 682GGATACATTT Deoxy, eeeekddddddddkeee sooosssssssssoss 9816 9832 1342CTACAGC MOE and cEt 684059 676 692 ATGTTTATCA Deoxy, eeekddddddddkeeeesoosssssssssooss 9826 9842 1348 GGATACA MOE and cEt 684064 676 692ATGTTTATCA Deoxy, eeeeddddddddkekee soosssssssssooss 9826 9842 1348GGATACA MOE and cEt 684068 676 692 ATGTTTATCA 4-8-5 eeeeddddddddeeeeesoosssssssssooss 9826 9842 1348 GGATACA MOE 684087 590 607 GGCGATCCCA5-8-5 eeeeeddddddddeeeee sooosssssssssooss 9740 9757 613 ATTACACC MOE684088 167 184 GTCGCCCTTC 5-8-5 eeeeeddddddddeeeee sooosssssssssooss 973990 1419 AGCACGCA MOE 684095 167 186 CCGTCGCCCT 5-10-5eeeeeddddddddddeeeee soooossssssssssooss 973 992 21 TCAGCACGCA MOE684097 167 186 CCGTCGCCCT 5-8-7 eeeeeddddddddeeeeeee sooossssssssssoooss973 992 21 TCAGCACGCA MOE 684101 588 607 GGCGATCCCA 6-8-6eeeeeeddddddddeeeeee sooossssssssssoooss 9738 9757 47 ATTACACCAC MOE684102 588 607 GGCGATCCCA 5-8-7 eeeeeddddddddeeeeeee sooossssssssssoooss9738 9757 47 ATTACACCAC MOE 684104 588 607 GGCGATCCCA 6-9-5eeeeeedddddddddeeeee soooossssssssssooss 9738 9757 47 ATTACACCAC MOE

The newly designed oligonucleotides were tested at various doses in A431cells. The modified oligonucleotides were tested in a series ofexperiments that had similar culture conditions. The results for eachexperiment are presented in separate tables shown below. Cells wereplated at a density of 5,000 cells per well and modifiedoligonucleotides were added to the media at 0.062 μM, 0.185 μM, 0.556μM, 1.667 μM, 5.000 μM, and 15.000 μM concentrations of modifiedoligonucleotide for free uptake by the cells, as specified in the Tablesbelow. After a treatment period of approximately 16 hours, RNA wasisolated from the cells and SOD-1 mRNA levels were measured byquantitative real-time PCR. Human primer probe sets RTS3898 was used tomeasure mRNA levels. SOD-1 mRNA levels were adjusted according to totalRNA content, as measured by RIBOGREEN®. Results are presented as percentinhibition of SOD-1, relative to untreated control cells.

TABLE 46 Dose response assay IC₅₀ ISIS No 0.062 μM 0.185 μM 0.556 μM1.667 μM 5.000 μM 15.000 μM (μM) 666846 18 28 45 70 69 81 0.8 666919 0 113 28 42 55 11.0 666849 33 29 52 62 74 82 0.6 666921 2 4 15 19 37 44 >15666853 20 29 49 69 76 83 0.7 666922 8 7 30 33 66 59 4.1 666859 26 30 5864 68 78 0.6 666861 6 21 44 76 68 77 1.1 666867 16 43 65 68 79 83 0.5666869 52 68 79 88 89 91 <0.06 666870 24 37 57 77 81 86 0.4

TABLE 47 Dose response assay IC₅₀ ISIS No 0.062 μM 0.185 μM 0.556 μM1.667 μM 5.000 μM 15.000 μM (μM) 684059 7 18 38 53 68 79 1.5 684102 0 90 0 4 0 >15 684064 12 19 29 38 51 61 5.0 684104 0 0 0 0 0 4 >15 684068 04 10 33 50 56 8.0 684087 3 1 29 0 0 27 >15 684088 10 11 11 3 4 18 >15684095 12 13 14 4 7 18 >15 684097 8 4 5 4 3 9 >15 684101 7 0 0 23 6 14>15

The newly designed oligonucleotides were also tested at various doses inSH-SY5Y cells. The modified oligonucleotides were tested in a series ofexperiments that had similar culture conditions. The results for eachexperiment are presented in separate tables shown below. Cells wereplated at a density of 20,000 cells per well and modifiedoligonucleotides transfected using electroporation at 0.062 μM, 0.185μM, 0.556 μM, 1.667 μM, 5.000 μM, and 15.000 μM concentrations ofmodified oligonucleotide, as specified in the Tables below. After atreatment period of approximately 16 hours, RNA was isolated from thecells and SOD-1 mRNA levels were measured by quantitative real-time PCR.Human primer probe set RTS3898 was used to measure mRNA levels. SOD-1mRNA levels were adjusted according to total RNA content, as measured byRIBOGREEN®. Results are presented as percent inhibition of SOD-1,relative to untreated control cells.

TABLE 48 Dose response assay IC₅₀ ISIS No 0.062 μM 0.185 μM 0.556 μM1.667 μM 5.000 μM 15.000 μM (μM) 666846 0 17 49 62 83 91 0.9 666919 10 323 35 77 78 2.2 666849 10 10 33 61 81 92 1.1 666921 0 0 12 30 56 68 4.8666853 0 17 39 59 85 82 1.3 666922 9 0 12 33 65 76 3.2 666859 11 44 5375 77 93 0.5 666861 0 0 34 61 81 90 1.4 666867 33 10 43 61 81 77 0.9666869 38 49 61 83 81 84 0.2 666870 3 6 48 69 77 87 1.1

TABLE 49 Dose response assay IC₅₀ ISIS No 0.062 μM 0.185 μM 0.556 μM1.667 μM 5.000 μM 15.000 μM (μM) 684059 4 30 51 68 88 92 0.7 684102 5 216 25 36 61 12.0 684064 15 27 52 63 79 92 0.7 684104 0 3 20 38 61 84 2.6684068 0 4 32 37 61 83 2.3 684087 0 3 21 31 47 66 5.8 684088 13 4 5 4052 77 3.9 684095 16 5 19 36 68 80 2.4 684097 11 15 9 30 59 76 3.6 6841010 0 8 23 49 66 6.6

Example 12: Inhibition of Human SOD-1 in a Transgenic Rat Model

Gapmers from the studies described above, including benchmark compoundISIS 333611, which was previously disclosed in WO 2005/040180, weretested in an SOD-1 transgenic rat model (Taconic, Cat#2148-F and2148-M). These hemizygous rats express mutant human SOD-1 in the spinalcord.

Additional gapmers were designed based on the sequences of theoligonucleotides disclosed in studies described above. Theoligonucleotides were designed as 5-9-5 MOE gapmers, 5-10-5 MOE gapmersor deoxy, MOE and cEt oligonucleotides. The 5-9-5 MOE gapmers are 19nucleosides in length, wherein the central gap segment is comprised ofnine 2′-deoxyribonucleosides and is flanked by wing segments on the 5′direction and the 3′ directions comprising five nucleosides each. The5-10-5 MOE gapmers are 20 nucleosides in length, wherein the central gapsegment is comprised of ten 2′-deoxyribonucleosides and is flanked bywing segments on the 5′ direction and the 3′ directions comprising fivenucleosides each. Each nucleoside in the 5′ wing segment and eachnucleoside in the 3′ wing segment has a 2′-MOE modification. The deoxy,MOE and cEt oligonucleotides are 17 nucleosides in length wherein eachnucleoside has a MOE sugar modification, a cEt sugar modification, or adeoxy moiety The sugar chemistry of each oligonucleotide is denoted asin the Chemistry column, where ‘k’ indicates a cEt modified sugar; ‘d’indicates a 2′-deoxyribose; and ‘e’ indicates a 2′-MOE modified sugar.The internucleoside linkages throughout each gapmer are eitherphosphodiester or phosphorothioate linkages. The internucleosidelinkages of each oligonucleotide is denoted in the Backbone Chemistrycolumn, where ‘o’ indicates a phosphodiester linkage and ‘s’ indicates aphosphorothioate linkage. All cytosine residues throughout eacholigonucleotide are 5-methylcytosines. “Start site” indicates the 5‘-most nucleoside to which the gapmer is targeted in the human genesequence. “Stop site” indicates the 3’-most nucleoside to which thegapmer is targeted human gene sequence. Each gapmer listed in the Tablebelow is targeted to either the human SOD-1 mRNA, designated herein asSEQ ID NO: 1 (GENBANK Accession No. NM_000454.4) or the human SOD-1genomic sequence, designated herein as SEQ ID NO: 2 (GENBANK AccessionNo. NT_011512.10 truncated from nucleotides 18693000 to Ser. No.18/704,000).

TABLE 50Modified oligonucleotides targeting human SOD-1 with mixed backbone chemistrySEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 1 NO: 2 NO: 2 ISIS Start StopBackbone Start Stop SEQ ID NO Site Site Sequence MotifSugar Modifications Chemistry Site Site NO 383872 167 186 CCGTCGCCCTT5-10-5 eeeeeddddddddddeeeee sooosssssssssssooos 973 992 21 CAGCACGCA MOE611457 165 184 GTCGCCCTTCA 5-10-5 eeeeeddddddddddeeeeesooosssssssssssooos 971 990 54 GCACGCACA MOE 611464 164 183 TCGCCCTTCAG5-10-5 eeeeeddddddddddeeeee sooosssssssssssooos 970 989 67 CACGCACAC MOE611467 656 675 TTTCTACAGCT 5-10-5 eeeeeddddddddddeeeeesooosssssssssssooos 9806 9825 272 AGCAGGATA MOE 611468 583 602TCCCAATTACA 5-10-5 eeeeeddddddddddeeeee sooosssssssssssooos 9733 9752227 CCACAAGCC MOE 611472 230 249 CCCCACACCTT 5-10-5 eeeeeddddddddddeeeeesooosssssssssssooos 4984 5003 145 CACTGGTCC MOE 611473 231 250TCCCCACACCT 5-10-5 eeeeeddddddddddeeeee sooosssssssssssooos 4985 5004146 TCACTGGTC MOE 611478 644 663 GCAGGATAAC 5-10-5 eeeeeddddddddddeeeeesooosssssssssssooos 9794 9813 260 AGATGAGTTA MOE 611479 645 664AGCAGGATAA 5-10-5 eeeeeddddddddddeeeee sooosssssssssssooos 9795 9814 261CAGATGAGTT MOE 611481 655 674 TTCTACAGCTA 5-10-5 eeeeeddddddddddeeeeesooosssssssssssooos 9805 9824 271 GCAGGATAA MOE 611484 660 679TACATTTCTAC 5-10-5 eeeeeddddddddddeeeee sooosssssssssssooos 9810 9829276 AGCTAGCAG MOE 611485 661 680 ATACATTTCTA 5-10-5 eeeeeddddddddddeeeeesooosssssssssssooos 9811 9830 277 CAGCTAGCA MOE 611488 124 143GCTAGGCCAC 5-10-5 eeeeeddddddddddeeeee sooosssssssssssooos 930 949 593GCCGAGGTCC MOE 611490 402 421 GTCAGCAGTCA 5-10-5 eeeeeddddddddddeeeeesooosssssssssssooos 8457 8476 666 CATTGCCCA MOE 611494 671 690GTTTATCAGGA 5-10-5 eeeeeddddddddddeeeee sooosssssssssssooos 9821 9840728 TACATTTCT MOE 611495 673 692 ATGTTTATCAG 5-10-5 eeeeeddddddddddeeeeesooosssssssssssooos 9823 9842 729 GATACATTT MOE 611498 569 588CAAGCCAAAC 5-10-5 eeeeeddddddddddeeeee sooosssssssssssooos 9719 9738 816GACTTCCAGC MOE 611499 664 683 AGGATACATTT 5-10-5 eeeeeddddddddddeeeeesooosssssssssssooos 9814 9833 822 CTACAGCTA MOE 612912 621 637CTCAGACTACA Deoxy, eeekkdddddddkkeee soosssssssssooss 9771 9787 1146TCCAAG MOE, and cEt 612915 656 672 CTACAGCTAGC Deoxy, eeekkdddddddkkeeesoosssssssssooss 9806 9822 1164 AGGATA MOE, and cEt 612917 684 700AGTGTTTAATG Deoxy, eeekkdddddddkkeee soosssssssssooss 9834 9850 1173TTTATC MOE, and cEt 612919 621 637 CTCAGACTACA Deoxy, eekkddddddddkkeeesoosssssssssooss 9771 9787 1146 TCCAAG MOE, and cEt 612923 656 672CTACAGCTAGC Deoxy, eekkddddddddkkeee soosssssssssooss 9806 9822 1164AGGATA MOE, and cEt 612924 674 690 GTTTATCAGGA Deoxy, eekkddddddddkkeeesoosssssssssooss 9824 9840 1346 TACATT MOE, and cEt 612934 170 186CCGTCGCCCTT Deoxy, eekkdddddddddkkee sooosssssssssoss 976 992 969 CAGCACMOE, and cEt 612935 585 601 CCCAATTACAC Deoxy, eekkdddddddddkkeesooosssssssssoss 9735 9751 1114 CACAAG MOE, and cEt 612940 659 675TTTCTACAGCT Deoxy, eekkdddddddddkkee sooosssssssssoss 9809 9825 1167AGCAGG MOE, and cEt 612942 668 684 CAGGATACATT Deoxy, eekkdddddddddkkeesooosssssssssoss 9818 9834 1344 TCTACA MOE, and cEt 612943 674 690GTTTATCAGGA Deoxy, eekkdddddddddkkee sooosssssssssoss 9824 9840 1346TACATT MOE, and cEt 666854 665 681 AGGATACATTT 5-9-5 eeeeedddddddddeeeeesooossssssssssooss 9815 9831 1428 CTACAGCT MOE 666855 666 682CAGGATACATT 5-9-5 eeeeedddddddddeeeee sooossssssssssooss 9816 9832 1461TCTACAGC MOE 666857 679 695 TTAATGTTTAT Deoxy, eeekddddddddkeeeesoosssssssssooss 9829 9845 1351 CAGGAT MOE, and cEt 666858 679 695TTAATGTTTAT Deoxy, eekkddddddddeeeee soosssssssssooss 9829 9845 1351CAGGAT MOE, and cEt 666864 679 695 TTAATGTTTAT Deoxy, kekeddddddddeeeeesoosssssssssooss 9829 9845 1351 CAGGAT MOE, and cEt 666865 679 695TTAATGTTTAT Deoxy, eeeeddddddddekeke soosssssssssooss 9829 9845 1351CAGGAT MOE, and cEt 666866 684 700 AGTGTTTAATG Deoxy, eeekddddddddkeeeesoosssssssssooss 9834 9850 1173 TTTATC MOE, and cEt 666908 686 702ACAGTGTTTAA Deoxy, eeeekdddddddkeeee sooossssssssooss 9836 9852 1175TGTTTA MOE, and cEt 666923 666 682 GGATACATTTC Deoxy, eeekddddddddkeeeesooossssssssooss 9816 9832 1342 TACAGC MOE, and cEt

The modified oligonucleotides were tested in a series of experimentsthat had similar conditions. The results for each experiment arepresented in separate tables shown below. Rats were injectedintrathecally with 304 of a 16.67 mg/ml solution of modifiedoligonucleotide diluted in PBS (500 μg final dose). A control group ofrats was injected intrathecally with PBS. Inhibition levels of SOD-1 inthe lumbar spinal cord, thoracic spinal cord and cervical spinal cordwere assessed. The data is presented below and indicate that severalmodified oligonucleotides inhibited human SOD-1 levels in this model.

TABLE 51 Percent inhibition of human SOD-1 in the spinal cord regions oftransgenic rats ISIS SEQ No Chemistry Lumbar Thoracic Cervical ID NO333611 5-10-5 MOE with 51 51 47 21 phosphorothioate backbone chemistry383872 5-10-5 MOE with 29 36 26 21 mixed backbone chemistry 6114605-10-5 MOE with 55 53 25 1428 mixed backbone chemistry 611464 5-10-5 MOEwith 52 54 26 67 mixed backbone chemistry 611468 5-10-5 MOE with 46 4419 227 mixed backbone chemistry 611481 5-10-5 MOE with 39 44 33 271mixed backbone chemistry

TABLE 52 Percent inhibition of human SOD-1 in the spinal cord regions oftransgenic rats ISIS SEQ No Chemistry Lumbar Thoracic Cervical ID NO611473 5-10-5 MOE with 47 42 5 146 mixed backbone chemistry 6114745-10-5 MOE with 75 65 65 149 mixed backbone chemistry 611479 5-10-5 MOEwith 24 13 20 261 mixed backbone chemistry 611484 5-10-5 MOE with 51 3141 276 mixed backbone chemistry 611485 5-10-5 MOE with 52 40 35 277mixed backbone chemistry 611492 5-10-5 MOE with 57 44 43 725 mixedbackbone chemistry

TABLE 53 Percent inhibition of human SOD-1 in the spinal cord regions oftransgenic rats ISIS SEQ No Chemistry Lumbar Thoracic Cervical ID NO611472 5-10-5 MOE with 0 19 15 145 mixed backbone chemistry 6114785-10-5 MOE with 16 33 24 260 mixed backbone chemistry 611490 5-10-5 MOEwith 53 55 44 666 mixed backbone chemistry 611494 5-10-5 MOE with 34 3938 728 mixed backbone chemistry 611495 5-10-5 MOE with 33 19 38 729mixed backbone chemistry 611498 5-10-5 MOE with 30 43 27 816 mixedbackbone chemistry 611499 5-10-5 MOE with 45 56 40 822 mixed backbonechemistry 611500 5-10-5 MOE with 56 58 52 823 mixed backbone chemistry

TABLE 54 Percent inhibition of human SOD-1 in the spinal cord regions oftransgenic rats ISIS SEQ No Chemistry Lumbar Thoracic Cervical ID NO611457 5-10-5 MOE with 56 46 43 54 mixed backbone chemistry 6114675-10-5 MOE with 21 28 22 272 mixed backbone chemistry 611488 5-10-5 MOEwith 14 23 4 593 mixed backbone chemistry 612917 Deoxy, MOE, and 47 5537 1173 cEt with mixed backbone chemistry 612923 Deoxy, MOE, and 53 6345 1164 cEt with mixed backbone chemistry 612925 Deoxy, MOE, and 67 6963 1348 cEt with mixed backbone chemistry 612928 Deoxy, MOE, and 84 8581 1351 cEt with mixed backbone chemistry

TABLE 55 Percent inhibition of human SOD-1 in the spinal cord regions oftransgenic rats ISIS SEQ No Chemistry Lumbar Thoracic Cervical ID NO612912 Deoxy, MOE, and cEt 59 60 48 1146 with mixed backbone chemistry612919 Deoxy, MOE, and cEt 60 60 58 1146 with mixed backbone chemistry612916 Deoxy, MOE, and cEt 72 69 69 1170 with mixed backbone chemistry612931 Deoxy, MOE, and cEt 81 79 72 1173 with mixed backbone chemistry612932 Deoxy, MOE, and cEt 21 26 24 1175 with mixed backbone chemistry

TABLE 56 Percent inhibition of human SOD-1 in the spinal cord regions oftransgenic rats ISIS SEQ No Chemistry Lumbar Thoracic Cervical ID NO612915 Deoxy, MOE, and cEt 54 48 52 1164 with mixed backbone chemistry612918 Deoxy, MOE, and cEt 73 69 64 1175 with mixed backbone chemistry612927 Deoxy, MOE, and cEt 82 75 62 1350 with mixed backbone chemistry612934 Deoxy, MOE, and cEt 59 44 48 969 with mixed backbone chemistry612935 Deoxy, MOE, and cEt 64 54 62 1114 with mixed backbone chemistry612940 Deoxy, MOE, and cEt 11 26 17 1167 with mixed backbone chemistry612941 Deoxy, MOE, and cEt 81 75 71 1342 with mixed backbone chemistry612942 Deoxy, MOE, and cEt 40 42 41 1344 with mixed backbone chemistry612943 Deoxy, MOE, and cEt 61 54 51 1346 with mixed backbone chemistry612944 Deoxy, MOE, and cEt 59 52 51 1348 with mixed backbone chemistry

TABLE 57 Percent inhibition of human SOD-1 in the spinal cord regions oftransgenic rats ISIS SEQ No Chemistry Lumbar Thoracic Cervical ID NO612924 Deoxy, MOE, and cEt 42 64 53 1346 with mixed backbone chemistry612947 Deoxy, MOE, and cEt 68 75 74 1351 with mixed backbone chemistry612948 Deoxy, MOE, and cEt 80 90 87 1352 with mixed backbone chemistry612949 Deoxy, MOE, and cEt 73 82 85 1172 with mixed backbone chemistry

TABLE 58 Percent inhibition of human SOD-1 in the spinal cord regions oftransgenic rats ISIS SEQ No Chemistry Lumbar Cervical ID NO 654304 5-8-5MOE with mixed 28 6 1429 backbone chemistry 654305 5-8-5 MOE with mixed14 0 1430 backbone chemistry 654306 5-8-5 MOE with mixed 36 0 1432backbone chemistry 654307 5-8-5 MOE with mixed 17 0 1432 backbonechemistry

TABLE 59 Percent inhibition of human SOD-1 in the spinal cord regions oftransgenic rats ISIS SEQ No Chemistry Lumbar Cervical ID NO 654334Deoxy, MOE, and cEt 39 19 1454 with mixed backbone chemistry 6668545-9-5 MOE with mixed 52 39 1428 backbone chemistry 666855 5-9-5 MOE withmixed 37 17 1461 backbone chemistry 666857 Deoxy, MOE, and cEt 59 391351 with mixed backbone chemistry 666858 Deoxy, MOE, and cEt 38 22 1351with mixed backbone chemistry 666859 Deoxy, MOE, and cEt 79 64 1351 withmixed backbone chemistry 666864 Deoxy, MOE, and cEt 50 40 1351 withmixed backbone chemistry 666865 Deoxy, MOE, and cEt 73 44 1351 withmixed backbone chemistry 666866 Deoxy, MOE, and cEt 67 56 1173 withmixed backbone chemistry 666908 Deoxy, MOE, and cEt 38 13 1175 withmixed backbone chemistry 666923 Deoxy, MOE, and cEt 45 26 1342 withmixed backbone chemistry

TABLE 60 Percent inhibition of human SOD-1 in the spinal cord regions oftransgenic rats ISIS SEQ No Chemistry Lumbar Cervical ID NO 654323Deoxy, MOE, and cEt 53 35 1433 with mixed backbone chemistry 6668465-10-5 MOE with mixed 64 50 725 backbone chemistry 666849 5-10-5 MOEwith mixed 63 55 725 backbone chemistry 666853 5-10-5 MOE with mixed 8174 725 backbone chemistry 666861 Deoxy, MOE, and cEt 55 47 1351 withmixed backbone chemistry 666867 Deoxy, MOE, and cEt 59 48 1173 withmixed backbone chemistry 666869 Deoxy, MOE, and cEt 82 81 1173 withmixed backbone chemistry 666870 Deoxy, MOE, and cEt 76 68 1173 withmixed backbone chemistry 666919 Deoxy, MOE, and cEt 76 68 1342 withmixed backbone chemistry 666921 Deoxy, MOE, and cEt 71 65 1342 withmixed backbone chemistry 666922 Deoxy, MOE, and cEt 67 62 1342 withmixed backbone chemistry

TABLE 61 Percent inhibition of human SOD-1 in the spinal cord regions oftransgenic rats ISIS SEQ No Chemistry Lumbar ID NO 684059 Deoxy, MOE,and cEt 54 1348 with mixed backbone chemistry 684064 Deoxy, MOE, and cEt51 1348 with mixed backbone chemistry 684068 4-8-5 MOE with mixed 181348 backbone chemistry 684087 5-8-5 MOE with mixed 37 613 backbonechemistry 684088 5-8-5 MOE with mixed 31 1419 backbone chemistry 6840955-10-5 MOE with mixed 34 21 backbone chemistry 684097 5-8-7 MOE withmixed 22 21 backbone chemistry 684101 6-8-6 MOE with mixed 22 47backbone chemistry 684104 6-9-5 MOE with mixed 11 47 backbone chemistry

Example 13: Dose-Dependent Inhibition of Human SOD-1 with ModifiedOligonucleotides in LLC-MK2 Cells

Gapmers from the studies described above, including benchmark compoundISIS 333611, exhibiting significant in vitro inhibition of SOD-1 mRNAwere selected and tested at various doses in LLC-MK2 cells. Thecross-reactivity of the human modified oligonucleotides tested in thisstudy with the rhesus monkey genomic sequence (the complement of GENBANKAccession No. NW_001114168.1 truncated from nucleotides 2258000 to2271000, designated herein as SEQ ID NO: 3) is shown in the Table below.

TABLE 62 Cross-reactivity of antisense oligonucleotides targeting humanSOD1with SEQ ID NO: 3 Start ISIS Site of SEQ No ID NO: 3 Mismatches333611 1572 2 436839 1564 0 436854 9049 0 436867 10347 0 666853 10375 1666859 10389 1 666919 10376 1 666921 10376 1

Cells were plated at a density of 20,000 cells per well and transfectedusing electroporation with 0.078 μM, 0.156 μM, 0.313 μM, 0.625 μM, 1.25μM, 2.50 μM, 5.00 μM, and 10.000 μM concentrations of modifiedoligonucleotide, as specified in the Tables below. After a treatmentperiod of approximately 16 hours, RNA was isolated from the cells andSOD-1 mRNA levels were measured by quantitative real-time PCR Primerprobe set RTS3121 (forward sequence TGGAGATAATACACAAGGCTGTACCA,designated herein as SEQ ID NO: 17; reverse sequenceCAACATGCCTCTCTTCATCCTTT, designated herein as SEQ ID NO: 18; probesequence ATCCTCTATCCAGACAACACGGTGGGC, designated herein as SEQ ID NO:19) was used to measure mRNA levels. SOD-1 mRNA levels were adjustedaccording to total RNA content, as measured by RIBOGREEN®. Results arepresented as percent inhibition of SOD-1, relative to untreated controlcells. The half maximal inhibitory concentration (IC₅₀) of eacholigonucleotide is also presented. As presented in the Table, several ofthe newly designed oligonucleotides were more potent than the benchmark,ISIS 336611.

TABLE 63 Dose-dependent inhibition of SOD-1 rhesus monkey mRNA IC₅₀ ISISNo 0.078 μM 0.156 μM 0.313 μM 0.625 μM 1.25 μM 2.50 μM 5.00 μM 10.00 μM(μM) 333611 3 2 0 0 17 15 19 40 >10 436839 0 0 5 0 20 22 37 61 7.1436854 34 34 40 39 52 65 69 84 1.2 436867 3 0 11 18 34 49 70 87 2.2666853 7 34 20 39 60 80 79 92 1.0 666859 0 9 20 18 15 25 30 44 >10666919 11 15 16 36 51 65 73 84 1.4 666921 0 13 28 37 50 52 62 74 1.8

Example 14: Tolerability of SOD-1 Modified Oligonucleotides in a RatModel

Gapmers from the studies described above, including benchmark compoundISIS 333611, which was previously disclosed in WO 2005/040180, weretested for tolerability in Sprague-Dawley rats.

The modified oligonucleotides were tested in a series of experimentsthat had similar conditions. Rats were injected intrathecally with 3 mgof a single dose of ISIS oligonucleotide. A control group of rats wasinjected intrathecally with PBS. Acute tolerability was assessed 3 hourspost-dose using a functional observational battery (FOB). This score isused to evaluate the acute tolerability of a compound with lower scoresdenoting better tolerated compounds. Control animals usually have ascore of ‘0’ or ‘1’. At 3 hours post injection, the rats are observed byplacing each rat on the cage top and evaluating certain functions,assigning a number of ‘0’ or ‘1’ depending on whether the rat exhibitsnormal function in the region of interest (0) or does not (1) for eachfunction, and then adding the total scores. Seven regions are assessed,including tail, hind paws, hind legs, hind end, front posture, forepaws, and head. The results of the scoring are presented in the Tablebelow. As presented in the Table, several newly designedoligonucleotides demonstrated more acute tolerability compared to thebenchmark, ISIS 333611.

TABLE 64 FOB scores in Sprague-Dawley rats Target Start ISIS Site on SEQFOB No ID NO: 1 Chemistry score 333611 167 5-10-5 MOE with 4phosphorothioate backbone 684073 167 Deoxy, MOE, and cEt 3 with mixedbackbone 684081 167 Deoxy, MOE, and cEt 1 with mixed backbone 684088 1675-8-5 MOE with mixed 0 backbone 684093 167 5-9-5 MOE with mixed 0backbone 684095 167 5-10-5 MOE with mixed 0 backbone 684096 167 6-8-6MOE with mixed 0 backbone 684097 167 5-8-7 MOE with mixed 0 backbone684098 167 7-8-5 MOE with mixed 0 backbone 684099 167 6-9-5 MOE withmixed 0 backbone 684074 168 Deoxy, MOE, and cEt 0 with mixed backbone684082 168 Deoxy, MOE, and cEt 1 with mixed backbone 684089 168 5-8-5MOE with mixed 0 backbone 684094 168 5-9-5 MOE with mixed 1 backbone684075 169 Deoxy, MOE, and cEt 3 with mixed backbone 684083 169 Deoxy,MOE, and cEt 3 with mixed backbone 684090 169 5-8-5 MOE with mixed 2backbone 684076 170 Deoxy, MOE, and cEt 2 with mixed backbone 684084 170Deoxy, MOE, and cEt 4 with mixed backbone 611474 234 5-10-5 MOE withmixed 4 backbone 654301 234 5-8-5 MOE with mixed 3 backbone 654302 2355-8-5 MOE with mixed 1 backbone 654303 236 5-8-5 MOE with mixed 0backbone 684069 588 Deoxy, MOE, and cEt 0 with mixed backbone 684077 588Deoxy, MOE, and cEt 1 with mixed backbone 684085 588 5-8-5 MOE withmixed 0 backbone 684091 588 5-9-5 MOE with mixed 0 backbone 684100 5885-10-5 MOE with mixed 0 backbone 684101 588 6-8-6 MOE with mixed 0backbone 684102 588 5-8-7 MOE with mixed 0 backbone 684103 588 7-8-5 MOEwith mixed 0 backbone 684104 588 6-9-5 MOE with mixed 0 backbone 684070589 Deoxy, MOE, and cEt 0 with mixed backbone 684078 589 Deoxy, MOE, andcEt 0 with mixed backbone 684086 589 5-8-5 MOE with mixed 0 backbone684092 589 5-9-5 MOE with mixed 0 backbone 684071 590 Deoxy, MOE, andcEt 0 with mixed backbone 684079 590 Deoxy, MOE, and cEt 0 with mixedbackbone 684087 590 5-8-5 MOE with mixed 0 backbone 684072 591 Deoxy,MOE, and cEt 1 with mixed backbone 684080 591 Deoxy, MOE, and cEt 1 withmixed backbone 654304 663 5-8-5 MOE with mixed 3 backbone 612916 664Deoxy, MOE, and cEt 0 with mixed backbone 654305 664 5-8-5 MOE withmixed 2 backbone 611492 665 5-10-5 MOE with mixed 0 backbone 654306 6655-8-5 MOE with mixed 3 backbone 654323 665 Deoxy, MOE, and cEt 0 withmixed backbone 654341 665 Deoxy, MOE, and cEt 0 with mixed backbone666846 665 5-10-5 MOE with mixed 0 backbone 666849 665 5-10-5 MOE withmixed 0 backbone 666851 665 5-10-5 MOE with mixed 1 backbone 666853 6655-10-5 MOE with mixed 0 backbone 666854 665 5-9-5 MOE with mixed 1backbone 611500 666 5-10-5 MOE with mixed 0 backbone 612941 666 Deoxy,MOE, and cEt 3 with mixed backbone 654307 666 5-8-5 MOE with mixed 2backbone 654342 666 Deoxy, MOE, and cEt 2 with mixed backbone 666845 6665-10-5 MOE with mixed 0 backbone 666848 666 5-10-5 MOE with mixed 1backbone 666850 666 5-10-5 MOE with mixed 0 backbone 666852 666 5-10-5MOE with mixed 1 backbone 666855 666 5-9-5 MOE with mixed 1 backbone666917 666 Deoxy, MOE, and cEt 3 with mixed backbone 666918 666 Deoxy,MOE, and cEt 3 with mixed backbone 666919 666 Deoxy, MOE, and cEt 2 withmixed backbone 666920 666 Deoxy, MOE, and cEt 1 with mixed backbone666921 666 Deoxy, MOE, and cEt 2 with mixed backbone 666922 666 Deoxy,MOE, and cEt 3 with mixed backbone 666923 666 Deoxy, MOE, and cEt 2 withmixed backbone 666856 667 5-9-5 MOE with mixed 3 backbone 612925 676Deoxy, MOE, and cEt 4 with mixed backbone 684059 676 Deoxy, MOE, and cEt4 with mixed backbone 684060 676 Deoxy, MOE, and cEt 3 with mixedbackbone 684061 676 Deoxy, MOE, and cEt 4 with mixed backbone 684062 676Deoxy, MOE, and cEt 4 with mixed backbone 684063 676 Deoxy, MOE, and cEt5 with mixed backbone 684064 676 Deoxy, MOE, and cEt 4 with mixedbackbone 684065 676 Deoxy, MOE, and cEt 4 with mixed backbone 684066 676Deoxy, MOE, and cEt 4 with mixed backbone 684067 676 Deoxy, MOE, and cEt5 with mixed backbone 684068 676 4-8-5 MOE with mixed 4 backbone 612927678 Deoxy, MOE, and cEt 4 with mixed backbone 654309 678 5-8-5 MOE withmixed 4 backbone 612928 679 Deoxy, MOE, and cEt 2 with mixed backbone612947 679 Deoxy, MOE, and cEt 7 with mixed backbone 654310 679 5-8-5MOE with mixed 3 backbone 666857 679 Deoxy, MOE, and cEt 1 with mixedbackbone 666858 679 Deoxy, MOE, and cEt 1 with mixed backbone 666859 679Deoxy, MOE, and cEt 1 with mixed backbone 666860 679 Deoxy, MOE, and cEt0 with mixed backbone 666861 679 Deoxy, MOE, and cEt 5 with mixedbackbone 666862 679 Deoxy, MOE, and cEt 1 with mixed backbone 666863 679Deoxy, MOE, and cEt 4 with mixed backbone 666864 679 Deoxy, MOE, and cEt4 with mixed backbone 666865 679 Deoxy, MOE, and cEt 5 with mixedbackbone 611497 681 5-10-5 MOE with mixed 5 backbone 612948 681 Deoxy,MOE, and cEt 3 with mixed backbone 666847 681 5-10-5 MOE with mixed 7backbone 612949 683 Deoxy, MOE, and cEt 4 with mixed backbone 612931 684Deoxy, MOE, and cEt 4 with mixed backbone 666866 684 Deoxy, MOE, and cEt6 with mixed backbone 666867 684 Deoxy, MOE, and cEt 6 with mixedbackbone 666868 684 Deoxy, MOE, and cEt 6 with mixed backbone 666869 684Deoxy, MOE, and cEt 6 with mixed backbone 666870 684 Deoxy, MOE, and cEt6 with mixed backbone 666871 684 Deoxy, MOE, and cEt 6 with mixedbackbone 666872 684 Deoxy, MOE, and cEt 6 with mixed backbone 666873 684Deoxy, MOE, and cEt 6 with mixed backbone 666874 684 Deoxy, MOE, and cEt5 with mixed backbone 612918 686 Deoxy, MOE, and cEt 4 with mixedbackbone 612932 686 Deoxy, MOE, and cEt 2 with mixed backbone 666906 686Deoxy, MOE, and cEt 2 with mixed backbone 666907 686 Deoxy, MOE, and cEt3 with mixed backbone 666908 686 Deoxy, MOE, and cEt 1 with mixedbackbone 666909 686 Deoxy, MOE, and cEt 0 with mixed backbone 666910 686Deoxy, MOE, and cEt 2 with mixed backbone 666911 686 Deoxy, MOE, and cEt0 with mixed backbone 666912 686 Deoxy, MOE, and cEt 0 with mixedbackbone 666913 686 Deoxy, MOE, and cEt 0 with mixed backbone 666914 686Deoxy, MOE, and cEt 0 with mixed backbone 666915 686 Deoxy, MOE, and cEt1 with mixed backbone 666916 686 Deoxy, MOE, and cEt 1 with mixedbackbone 654318 687 5-8-5 MOE with mixed 1 backbone 654334 687 Deoxy,MOE, and cEt 3 with mixed backbone

Tolerability was also assessed 8 weeks post-dose by measuring the levelsof IBA1, a microglial marker, and GFAP, an astrocytic marker, in thelumbar spinal cord region. Both IBA1 and GFAP are markers of CNSinflammation (Frank, M G, Brain Behav. Immun. 2007, 21, 47-59), hencethe higher the level of either marker, the less tolerable the antisenseoligonucleotide is deemed to be in this rat model.

IBA1 mRNA levels were measured with primer probe set rAIF1_LTS00219(forward sequence AGGAGAAAAACAAAGAACACCAGAA, designated herein as SEQ IDNO: 5; reverse sequence CAATTAGGGCAACTCAGAAATAGCT, designated herein asSEQ ID NO: 6; probe sequence CCAACTGGTCCCCCAGCCAAGA, designated hereinas SEQ ID NO: 7). GFAP mRNA levels were measured with primer probe setmGFAP_LTS00370 (forward sequence GAAACCAGCCTGGACACCAA, designated hereinas SEQ ID NO: 8; reverse sequence TCCACAGTCTTTACCACGATGTTC, designatedherein as SEQ ID NO: 9; probe sequence TCCGTGTCAGAAGGCCACCTCAAGA,designated herein as SEQ ID NO: 10).

The results are presented in the Table below. As presented in the Table,several newly designed oligonucleotides were more tolerable compared tothe benchmark, ISIS 333611.

TABLE 65 IBA1 and GFAP mRNA levels (% control) in the lumbar regions ofSprague-Dawley rats ISIS No. IBA1 GFAP 333611 341 314 654301 149 137654302 261 129 654303 110 80 654304 143 130 654305 185 158 654306 110106 654307 152 144 654309 195 169 654310 119 141 654318 93 81 654323 125113 654334 114 75 654341 209 224 654342 473 485 666845 389 416 666846173 171 666847 271 297 666848 399 377 666849 140 150 666850 246 252666851 246 199 666852 282 266 666853 168 147 666854 135 123 666855 238221 666856 253 209 666857 242 182 666858 169 134 666859 185 162 666861161 152 666862 254 285 666863 216 185 666864 174 154 666865 251 232666866 281 135 666867 132 112 666868 199 211 666869 262 207 666870 201189 666871 192 214 666872 441 136 666873 340 277 666874 204 199 666917292 244 666919 115 85 666920 155 102 666921 108 82 666922 123 82 666923118 93 684059 168 162 684060 158 141 684061 335 263 684062 218 265684064 191 168 684065 245 304 684066 313 376 684067 171 151 684068 157135 684085 459 586 684086 187 227 684087 215 263 684088 151 183 684089507 667 684090 130 170 684091 350 426 684092 366 333 684093 412 264684094 294 373 684095 213 215 684096 404 335 684097 217 206 684098 378438 684099 534 473 684100 276 259 684101 153 125 684102 237 242 684103588 416 684104 221 193

Example 15: Dose Dependent Inhibition of Human SOD-1 in a Transgenic RatModel

Gapmers from the studies described above, including benchmark compoundISIS 333611, were tested in an SOD-1 transgenic rat model (Taconic,Cat#2148-F and 2148-M). These hemizygous rats express mutant human SOD-1in the spinal cord, many brain regions, and peripheral organs.

Rats were injected intrathecally with 10, 30, 100, 300, 1000, or 3000 μgof a gapmer listed in the table below or with only PBS. Two weeks later,the animals were sacrificed. Inhibition of SOD-1 mRNA in the lumbarspinal cord, cervical spinal cord, rostral cortex, and caudal cortex wasassessed by RT-PCR using primer probe set RTS3898, described inExample 1. The data is presented below as ED₅₀ values, and indicatesthat the oligonucleotides inhibited SOD1 mRNA in multiple CNS tissuesmore potently than Isis 333611. Indeed, ED₅₀ values for Isis No. 333611could not even be calculated, as indicated by an entry of “n/a,” becauseeven the highest concentration tested (3000 μg) did not inhibit SOD-1mRNA greater than 55-65%. “n.d.” indicates that there is no dataavailable for the indicated sample.

TABLE 66 Inhibition of human SOD1 in transgenic rats Isis ED₅₀ (μg) SEQNo. Lumbar Cervical Rostral Caudal ID NO. 333611 n/a n/a n.d. n.d. 21666853 81.3 242.6 6434 931 725 666859 74.0 358.8 2360 1113 1351 666870139.4 1111 5511 2105 1173 666919 104.1 613.5 >6000 2655 1342

Example 16: Tolerability of SOD-1 Modified Oligonucleotides in Rats

Gapmers from the studies described above, including benchmark compoundISIS 333611, were tested for tolerability in Sprague-Dawley rats. Groupsof 4 to 6 rats were injected intrathecally with 1 mg or 3 mg of a singledose of an ISIS oligonucleotide. A control group of rats was injectedintrathecally with PBS. Acute tolerability was assessed 3 hourspost-dose, as described in Example 14. The results for the 1 mg dose arethe averages for each group following one experiment. The results forthe 3 mg dose are the averages for each group across two replicateexperiments. The results of the study, presented in the table below,indicate that several newly designed oligonucleotides were moretolerable than the benchmark, ISIS 333611.

TABLE 67 FOB values Isis 3 hour FOB 8 week FOB No. 1 mg 3 mg 1 mg 3mg333611 3.0 4.9 0.0 1.2 666853 0.0 0.5 0.0 0.0 666859 0.0 2.1 0.0 0.3666870 2.3 5.8 0.0 0.8 666919 1.3 3.5 0.0 0.1

Example 17: Dose Dependent Inhibition of Human SOD-1 in a TransgenicMouse Model

In order to confirm the results obtained in transgenic rats in anotherspecies, gapmers from the studies described above were tested in anSOD-1 transgenic mouse model that expresses the same G93A human mutantSOD1 gene that the transgenic rat expresses (see Examples 12 and 15).

Mice received an intracerebral ventricular bolus (ICVB) of 10, 30, 100,300, or 700 μg of a gapmer listed in the table below, or PBS. Two weekslater, the animals were sacrificed. Inhibition of SOD-1 mRNA in thelumbar spinal cord and cortex was assessed by RT-PCR using primer probeset RTS3898, described in Example 1. The data is presented below as ED₅₀values, and indicates that the oligonucleotides inhibited SOD1 mRNA morepotently than Isis 333611 in both rats and mice.

TABLE 68 Inhibition of human SOD1 in transgenic mice Isis Lumbar ED₅₀Cortex ED₅₀ No. (μg) (μg) 333611 401 786 666853 136 188 666859 106 206666870 148 409 666919 168 1211

Example 18: Tolerability of SOD-1 Modified Oligonucleotides in Mice

Gapmers from the studies described above, including benchmark compoundISIS 333611, were tested for tolerability in C57bl6 mice. Mice wereinjected stereotaxically into the cerebral ventricles with 700 ug of asingle dose of ISIS oligonucleotide. A control group of mice wasinjected into the cerebral ventricle with PBS. Acute tolerability wasassessed at 3 hours post injection using a functional observationbattery (FOB) different from that used for the rats. Each mouse wasevaluated according to 7 different criteria. The 7 criteria are (1) themouse was bright, alert, and responsive; (2) the mouse was standing orhunched without stimuli; (3) the mouse shows any movement withoutstimuli (4) the mouse demonstrates forward movement after its lifted;(5) the mouse demonstrates any movement after its lifted; (6) the mouseresponds to a tail pinch; (7) regular breathing. For each of the 7different criteria, each mouse was given a sub-score of 0 if it met thecriteria or 1 if it did not. After all of the 7 criteria were evaluated,the sub-scores were summed for each mouse and then averaged for eachgroup. For example, if a mouse was bright, alert, and responsive 3 hoursafter the 700 μg ICV dose, and met all other criteria, it would get asummed score of 0. If another mouse was not bright, alert, andresponsive 3 hours after the 700 μg ICV dose but met all other criteria,it would receive a score of 1. Saline treated mice generally receive ascore of 0. A score at the top end of the range would be suggestive ofacute toxicity.

Body weights were measured throughout the study and are reported belowas percent change at 8 weeks relative to baseline. Long termtolerability was assessed 8 weeks post-dose by measuring the levels ofIBA1 and GFAP, as described in Example 14. IBA1 and GFAP mRNA levels arereported relative to PBS treated animals. The results of the study,presented in the tables below, indicate that several newly designedoligonucleotides were more tolerable, in rats and mice, compared to thebenchmark, ISIS 333611.

TABLE 69 FOB values and body weight change Isis Body weight No. 3 hourFOB (% change) 333611 6.5 3.8 666853 1.25 8.0 666859 1.75 14.0 6668704.75 7.3 666919 0.0 5.2

TABLE 70 Inflammation markers Isis IBA1 (% PBS) GFAP (% PBS) No. LumbarCortex Lumbar Cortex 333611 130.3 134.3 117.5 207.7 666853 102.8 109.3103.3 103.7 666859 110.4 98.2 109.0 72.8 666870 158.8 117.8 106.7 128.6666919 115.0 97.9 99.8 84.3

Example 19: Dose Dependent Inhibition of Monkey SOD-1 in CynomolgusMonkey

Isis No. 666853 was tested in cynomolgus monkey. There is one mismatchbetween Isis No. 666853 and cynomolgus monkey SOD-1, and there are 17contiguous bases in Isis No. 666853 that are 100% complementary tocynomolgus monkey SOD-1.

Groups of 6-10 male and female monkeys received an intrathecal lumbarbolus of PBS or 4, 12, or 35 mg of Isis No. 666853 on days 1, 14, 28,56, and 84 of the study. Each group received the same dose on all fivedosing days. On day 91, the animals were sacrificed. Inhibition of SOD-1mRNA in the lumbar, thoracic, and cervical spinal cord and frontalcortex, motor cortex, hippocampus, pons, and cerebellum was assessed byRT-PCR using primer probe set RTS3898. The data is presented below asthe average percent inhibition for each treatment group, relative to thePBS treated group. The results indicate that Isis No. 666853 inhibitedSOD-1 mRNA in multiple target tissues in cynomolgus monkey.

Treatment with 666853 was well tolerated for the duration of the 13 weekstudy and there were no clinical observations of adverse reactions inmonkeys.

TABLE 71 Inhibition of SOD-1 mRNA in monkeys Amount of 666853 Inhibition(%) per dose Frontal Motor (mg) Lumbar Thoracic Cervical cortex cortexHippocampus Pons Cerebellum 4 44.4 27.1 20.1 21.5 21.6 32.0 6.8 15.4 1275.4 69.0 42.1 56.7 55.7 31.8 13.2 33.3 35 87.0 74.8 72.1 80.5 82.6 80.148.6 48.4

What is claimed is:
 1. A salt of an antisense compound, wherein theanion of the antisense compound has the following formula:


2. An antisense compound according to the following formula: mCes AeoGes Geo Aes Tds Ads mCds Ads Tds Tds Tds mCds Tds Ads mCeo Aes Geo mCesTe (nucleobase sequence of SEQ ID NO: 725); wherein A=an adenine, mC=a5-methylcytosine, G=a guanine, T=a thymine, e=a 2′-O-methoxyethylribosemodified sugar, d=a 2′-deoxyribose sugar, s=a phosphorothioateinternucleoside linkage, and o=a phosphodiester internucleoside linkage,or a salt thereof.
 3. A composition comprising the antisense compound orsalt thereof of claim 2 and at least one of a pharmaceuticallyacceptable carrier or diluent.
 4. A composition comprising the salt ofthe antisense compound of claim 1 and at least one of a pharmaceuticallyacceptable carrier or diluent.
 5. The composition of claim 3, whereinthe pharmaceutically acceptable carrier or diluent is phosphate-bufferedsaline (PBS).
 6. The composition of claim 4, wherein thepharmaceutically acceptable carrier or diluent is PBS.
 7. Thecomposition of claim 3, which is formulated for intrathecaladministration.
 8. The composition of claim 4, which is formulated forintrathecal administation.
 9. The antisense compound of claim
 2. 10. Thesalt of the antisense compound of claim
 2. 11. A composition comprisingthe antisense compound of claim 9 and at least one of a pharmaceuticallyacceptable carrier or diluent.
 12. The composition of claim 11, whereinthe pharmaceutically acceptable carrier or diluent is PBS.
 13. Thecomposition of claim 11, which is formulated for intrathecaladministration.
 14. A composition comprising the salt of the antisensecompound of claim 10 and at least one of a pharmaceutically acceptablecarrier or diluent.
 15. The composition of claim 14, wherein thepharmaceutically acceptable carrier or diluent is PBS.
 16. Thecomposition of claim 14, which is formulated for intrathecaladministration.
 17. An antisense compound according to the followingformula:

or a salt thereof.
 18. A composition comprising the antisense compoundor salt thereof of claim 17 and at least one of a pharmaceuticallyacceptable carrier or diluent.
 19. The composition of claim 18, whereinthe pharmaceutically acceptable carrier or diluent is PBS.
 20. Thecomposition of claim 18, which is formulated for intrathecaladministration.
 21. The antisense compound of claim
 17. 22. Acomposition comprising the antisense compound of claim 21 and at leastone of a pharmaceutically acceptable carrier or diluent.
 23. Thecomposition of claim 22, wherein the pharmaceutically acceptable carrieror diluent is PBS.
 24. The composition of claim 22, which is formulatedfor intrathecal administration.